Il-17a/f heterologous polypeptides and therapeutic uses thereof

ABSTRACT

The present invention is directed to a novel naturally occurring human cytokine that is comprised of a heterodimer of interleukin-17 and interleukin-17F designated herein as interleukin 17A/F (IL-17A/F). Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, specific antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided herein are methods for treating degenerative cartilaginous disorders and other inflammatory diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/860,824, filed Jun. 2, 2004, which is a non-provisionalapplication filed under 37 CFR 1.53(b), claiming priority under 35 USCSection 119(e) to Provisional Application Nos. 60/486,457 filed on Jul.11, 2004 and 60/485,599 filed on Jul. 8, 2003, the entire disclosures ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of a novel human cytokine designated herein asinterleukin-17A/F (IL-17A/F).

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents.

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Similarly to secreted proteins, membrane-bound proteins and receptormolecules have various industrial applications, including aspharmaceutical and diagnostic agents. Receptor immunoadhesins, forinstance, can be employed as therapeutic agents to block receptor-ligandinteractions. The membrane-bound proteins can also be employed forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native secreted proteins and native receptor or membrane-boundproteins. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted proteins. Examples of screening methods and techniques aredescribed in the literature [see, for example, Klein et al., Proc. Natl.Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

In this regard, the present invention relates to identifying novelsecreted polypeptides of the interleukin-17 (IL-17) family which havebeen shown to be related to immune-mediated and inflammatory disease.Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

A central event in both humoral and cell mediated inunune responses isthe activation and clonal expansion of helper T cells. Helper T cellactivation is initiated by the interaction of the T cell receptor(TCR)-CD3 complex with an antigen-MHC on the surface of an antigenpresenting cell. This interaction mediates a cascade of biochemicalevents that induce the resting helper T cell to enter a cell cycle (theGO to G1 transition) and results in the expression of a high affinityreceptor for IL-2 and sometimes IL-4. The activated T cell progressesthrough the cycle proliferating and differentiating into memory cells oreffector cells.

In addition to the signals mediated through the TCR, activation of Tcells involves additional costimulation induced by cytokines released bythe antigen presenting cell or through interactions with membrane boundmolecules on the antigen presenting cell and the T cell. The cytokinesIL-1 and IL-6 have been shown to provide a costimulatory signal. Also,the interaction between the B7 molecule expressed on the surface of anantigen presenting cell and CD28 and CTLA-4 molecules expressed on the Tcell surface effect T cell activation. Activated T cells express anincreased number of cellular adhesion molecules, such as ICAM-1,integrins, VLA-4, LFA-1, CD56, etc.

T-cell proliferation in a mixed lymphocyte culture or mixed lymphocytereaction (MLR) is an established indication of the ability of a compoundto stimulate the immune system. In many immune responses, inflammatorycells infiltrate the site of injury or infection. The migrating cellsmay be neutrophilic, eosinophilic, monocytic or lymphocytic as can bedetermined by histologic examination of the affected tissues. CurrentProtocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,Inc.

Immune related diseases could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

Interleukin-17 (IL-17) is a T-cell derived pro-inflammatory moleculethat stimulates epithelial, endothelial and fibroblastic cells toproduce other inflammatory cytokines and chemokines including IL-6,IL-8, G-CSF, and MCP-1 [see, Yao, Z. et al., J. Immunol.,122(121:5483-5486 (1995); Yao, Z. et al., Immunity, 3(6:811-821 (1995);Fossiez, F., et al., J. Exp. Med., 183(6): 2593-2603 (1996); Kennedy,J., et al., J. Interferon Cytokine Res., 16(8):611-7 (1996); Cai, X. Y.,et al., Immunol. Lett, 62(1):51-8 (1998); Jovanovic, D. V., et al., J.Immunol., 160(7):3513-21 (1998); Laan, M., et al., J. Immunol.,162(4):2347-52 (1999); Linden, A., et al., Eur Respir J, 15(5):973-7(2000); and Aggarwal, S. and Gurney, A. L., J Leukoc Biol, 71(1):1-8(2002)]. IL-17 also synergizes with other cytokines including TNF-α andIL-1β to further induce chemokine expression (Chabaud, M., et al., J.Immunol. 161(1):409-14 (1998)). Interleukin 17 (IL-17) exhibitspleitropic biological activities on various types of cells. IL-17 alsohas the ability to induce ICAM-1 surface expression, proliferation of Tcells, and growth and differentiation of CD34⁺ human progenitors intoneutrophils. IL-17 has also been implicated in bone metabolism, and hasbeen suggested to play an important role in pathological conditionscharacterized by the presence of activated T cells and TNF-α productionsuch as rheumatoid arthritis and loosening of bone implants (VanBezooijen et al., J. Bone Miner. Res., 14: 1513-1521 [1999]). ActivatedT cells of synovial tissue derived from rheumatoid arthritis patientswere found to secrete higher amounts of IL-17 than those derived fromnormal individuals or osteoarthritis patients (Chabaud et al., ArthritisRheum., 42: 963-970 [1999]). It was suggested that this proinflammatorycytokine actively contributes to synovial inflammation in rheumatoidarthritis. Apart from its proinflammatory role, IL-17 seems tocontribute to the pathology of rheumatoid arthritis by yet anothermechanism. For example, IL-17 has been shown to induce the expression ofosteoclast differentiation factor (ODF) mRNA in osteoblasts (Kotake etal., J. Clin. Invest., 103: 1345-1352 [1999]). ODF stimulatesdifferentiation of progenitor cells into osteoclasts, the cells involvedin bone resorption. Since the level of IL-17 is significantly increasedin synovial fluid of rheumatoid arthritis patients, it appears thatIL-17 induced osteoclast formation plays a crucial role in boneresorption in rheumatoid arthritis. IL-17 is also believed to play a keyrole in certain other autoimmune disorders such as multiple sclerosis(Matusevicius et al., Mult. Scler., 5: 101-104 (1999); Kurasawa, K., etal., Arthritis Rheu 43(11):2455-63 (2000)) and psoriasis (Teunissen, M.B., et al., J Invest Dermatol 111(4):645-9 (1998); Albanesi, C., et al.,J Invest Dermatol 115(1):81-7 (2000); and Homey, B., et al., J. Immunol.164(12:6621-32 (2000)).

IL-17 has further been shown, by intracellular signalling, to stimulateCa²⁺ influx and a reduction in [cAMP]_(i) in human macrophages(Jovanovic et al., J. Immunol., 160:3513 [1998]). Fibroblasts treatedwith IL-17 induce the activation of NF-κB, [Yao et al., Immunity, 3:811(1995), Jovanovic et al., supra], while macrophages treated with itactivate NF-κB and mitogen-activated protein kinases (Shalom-Barek etal., J. Biol. Chem., 273:27467 [1998]). Additionally, IL-17 also sharessequence similarity with mammalian cytokine-like factor 7 that isinvolved in bone and cartilage growth. Other proteins with which IL-17polypeptides share sequence similarity are human embryo-derivedinterleukin-related factor (EDIRF) and interleukin-20.

Consistent with IL-17's wide-range of effects, the cell surface receptorfor IL-17 has been found to be widely expressed in many tissues and celltypes (Yao et al., Cytokine, 9:794 [1997]). While the amino acidsequence of the human IL-17 receptor (IL-R) (866 amino acids) predicts aprotein with a single transmembrane domain and a long, 525 amino acidintracellular domain, the receptor sequence is unique and is not similarto that of any of the receptors from the cytokine/growth factor receptorfamily. This coupled with the lack of similarity of IL-17 itself toother known proteins indicates that IL-17 and its receptor may be partof a novel family of signaling proteins and receptors. It has beendemonstrated that IL-17 activity is mediated through binding to itsunique cell surface receptor (designated herein as human IL-17R),wherein previous studies have shown that contacting T cells with asoluble form of the IL-17 receptor polypeptide inhibited T cellproliferation and IL-2 production induced by PHA, concanavalin A andanti-TCR monoclonal antibody (Yao et al., J. Immunol., 155:5483-5486[1995]). As such, there is significant interest in identifying andcharacterizing novel polypeptides having homology to the known cytokinereceptors, specifically IL-17 receptors.

Interleukin 17 is now recognized as the prototype member of an emergingfamily of cytokines. The large scale sequencing of the human and othervertebrate genomes has revealed the presence of additional genesencoding proteins clearly related to IL-17, thus defining a new familyof cytokines. There are at least 6 members of the IL-17 family in humansand mice including IL-17B, IL-17C, IL-17D, IL-17E and IL-17F as well asnovel receptors IL-17RH1, IL-17RH2, IL-17RH3 and IL-17RH4 (seeWO01/46420 published Jun. 28, 2001). One such IL-17 member (designatedas IL-17F) has been demonstrated to bind to the human IL-17 receptor(IL-17R) (Yao et al., Cytokine, 9(11):794-800 (1997)). Initialcharacterization suggests that, like IL-17, several of these newlyidentified molecules have the ability to modulate immune function. Thepotent inflammatory actions that have been identified for several ofthese factors and the emerging associations with major human diseasessuggest that these proteins may have significant roles in inflammatoryprocesses and may offer opportunities for therapeutic intervention.

The gene encoding human IL-17F is located adjacent to IL-17 (Hymowitz,S. G., et al., Embo J, 20(19):5332-41 (2001)). IL-17 and IL-17F share44% amino acid identity whereas the other members of the IL-17 familyshare a more limited 15-27% amino acid identity suggesting that IL-17and IL-17F form a distinct subgroup within the IL-17 family (Starnes,T., et al., J Immunol, 167(8):4137-40 (2001); Aggarwal, S. and Gurney,A. L., J. Leukoc Biol 71(1):1-8 (2002)). IL-17F appears to have similarbiological actions as IL-17, and is able to promote the production ofIL-6, IL-8, and G-CSF from a wide variety of cells. Similar to IL-17, itis able to induce cartilage matrix release and inhibit new cartilagematrix synthesis (see US-2002-0177188-A1 published Nov. 28, 2002). Thus,like IL-17, IL-17F may potentially contribute to the pathology ofinflammatory disorders. Recently, these authors have observed that bothIL-17 and IL-17F are induced in T cells by the action of interleukin 23(IL-23) (Aggarwal, S., et al., J. Biol. Chem., 278(3):1910-4 (2003)).The observation that IL-17 and IL-17F share similar chromosomallocalization and significant sequence similarity sd well as theobservation that IL-17 and IL-17F appear to be induced with the samecell population in response to a specific stimuli has lead to theidentification of a new human cytokine that is comprised of a covalentheterodimer of IL-17 and IL-17F (herein designated IL-17A/F). HumanIL-17A/F is a distinctly new cytokine, distinguishable from human IL-17and IL-17F in both protein structure and in cell-based activity assays.Through the use of purified recombinant human IL-17A/F as a standard, ahuman IL-17AF-specific ELISA has been developed. Through the use of thisspecific ELISA, the induced expression of human IL-17A/F was detected,confirming that IL-17A/F is naturally produced from activated human Tcells in culture. Hence, IL-17A/F is a distinctly new cytokine,detectable as a natural product of isolated activated human T cells,whose recombinant form has been characterized, in both protein structureand cell-based assays, as to be different and distinguishable fromrelated cytokines. Thus, these studies provide and identify a novelimmune stimulant (i.e. IL-17A/F) that can boost the immune system torespond to a particular antigen that may not have been immunologicallyactive previously. As such, the newly identified immune stimulant hasimportant clinical applications. This novel IL-17A/F cytokine oragonists thereof, would therefore find practical utility as an immunestimulant, whereas molecules which inhibit IL-17A/F activity(antagonists) would be expected to find practical utility when aninhibition of the immune response is desired, such as in autoimmunediseases. Specifically, antibodies to this new cytokine which eithermimic (agonist antibodies) or inhibit (antagonist antibodies) theimmunological activities of IL-17A/F would possess therapeuticqualities. Small molecules which act to inhibit the activity of thisnovel cytokine would also have potential therapeutic uses.

SUMMARY OF THE INVENTION A. Embodiments

The present invention concerns compositions and methods useful for thediagnosis and treatment of immune related disease in mammals, includinghumans. The present invention is based on the identification of proteins(including agonist and antagonist antibodies) which either stimulate orinhibit the immune response in mammals. Immune related diseases can betreated by suppressing or enhancing the immune response. Molecules thatenhance the immune response stimulate or potentiate the immune responseto an antigen. Molecules which stimulate the immune response can be usedtherapeutically where enhancement of the immune response would bebeneficial. Alternatively, molecules that suppress the immune responseattenuate or reduce the immune response to an antigen (e.g.,neutralizing antibodies) can be used therapeutically where attenuationof the immune response would be beneficial (e.g., inflammation).Accordingly, the IL-17A/F polypeptides of the present invention andagonists and antagonists thereof are also useful to prepare medicinesand medicaments for the treatment of immune-related and inflammatorydiseases. In a specific aspect, such medicines and medicaments comprisea therapeutically effective amount of an IL-17A/F polypeptide, agonistor antagonist thereof with a pharmaceutically acceptable carrier.Preferably, the admixture is sterile.

In a further embodiment, the invention concerns a method of identifyingagonists of or antagonists to an IL-17A/F polypeptide which comprisescontacting the IL-17A/F polypeptide with a candidate molecule andmonitoring a biological activity mediated by said IL-17A/F polypeptide.Preferably, the IL-17A/F polypeptide is a native sequence IL-17A/Fpolypeptide. In a specific aspect, the IL-17A/F agonist or antagonist isan anti-IL-17A/F antibody.

In another embodiment, the invention concerns a composition of mattercomprising an IL-17A/F polypeptide or an agonist or antagonist antibodywhich binds the polypeptide in admixture with a carrier or excipient. Inone aspect, the composition comprises a therapeutically effective amountof the polypeptide or antibody. In another aspect, when the compositioncomprises an immune stimulating molecule, the composition is useful for:(a) enhancing infiltration of inflammatory cells into a tissue of amammal in need thereof, (b) stimulating or enhancing an immune responsein a mammal in need thereof, (c) increasing the proliferation ofT-lymphocytes in a mammal in need thereof in response to an antigen, (d)stimulating the activity of T-lymphocytes or (e) increasing the vascularpermeability. In a further aspect, when the composition comprises animmune inhibiting molecule, the composition is useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of T-lymphocytes or(d) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncomprises a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

In another embodiment, the invention concerns a method of treating animmune related disorder in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of anIL-17A/F polypeptide, an agonist thereof, or an antagonist thereto. In apreferred aspect, the immune related disorder is selected form the groupconsisting of: systemic lupus erythematosis, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis, idiopathic inflammatory myopathies, Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, thyroiditis, diabetes mellitus,immune-mediated renal disease, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious, autoimmune chronic active hepatitis, primary biliarycirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases such as asthma, allergic rhinitis, atopic dermatitis,food hypersensitivity and urticaria, immunologic diseases of the lungsuch as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In one aspect, the presentinvention concerns an isolated antibody which binds an IL-17A/Fpolypeptide. In another aspect, the antibody mimics the activity of anIL-17A/F polypeptide (an agonist antibody) or conversely the antibodyinhibits or neutralizes the activity of an IL-17A/F polypeptide (anantagonist antibody). In another aspect, the antibody is a monoclonalantibody, which preferably has nonhuman complementarity determiningregion (CDR) residues and human framework region (FR) residues. Theantibody may be labeled and may be immobilized on a solid support. In afurther aspect, the antibody is an antibody fragment, a monoclonalantibody, a single-chain antibody, or an anti-idiotypic antibody. Inanother aspect, the antibody fragment or single-chain antibody comprisesa Fab fragment selected from the group consisting of the amino acidsequence shown in FIG. 6 as SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ TD NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42, wherein said Fab fragment further comprises three heavy chainvariable regions containing CDR-H1 consisting of amino acid residues 7to 16 of SEQ ID NOs:9-42, CDR-H2 consisting of amino acid residues 30 to46 of SEQ ID NOs:9-42, and CDR-H3 consisting of amino acid residue 78 toat least amino acid residue 96 of SEQ ID NOs:9-42, wherein said Fabfragment is capable of binding IL-17A/F. In another aspect, the antibodyfragment or single-chain antibody comprises a Fab fragment selected fromthe group consisting of the amino acid sequence shown in FIG. 6 as SEQID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, wherein said Fabfragment further comprises at least heavy chain variable regioncontaining CDR-H1 consisting of amino acid residues 7 to 16 of SEQ IDNOs:9-42, and CDR-H2 consisting of amino acid residues 30 to 46 of SEQID NOs:9-42, wherein said Fab fragment is capable of binding IL-17A/F.In another aspect, the antibody fragment or single-chain antibodycomprises a Fab fragment selected from the group consisting of the aminoacid sequence shown in FIG. 6 as SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, and SEQ ID NO:42, wherein said Fab fragment further comprises atleast heavy chain variable regions containing CDR-H1 consisting of aminoacid residues 7 to 16 of SEQ ID NOs:9-42 and CDR-H3 consisting of aminoacid residue 78 to at least amino acid residue 96 of SEQ ID NOs:9-42,wherein said Fab fragment is capable of binding IL-17A/F. In anotheraspect, the antibody fragment or single-chain antibody comprises a Fabfragment selected from the group consisting of the amino acid sequenceshown in FIG. 6 as SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ 1D NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42, wherein said Fab fragment further comprises at least heavychain variable regions containing CDR-H2 consisting of amino acidresidues 30 to 46 of SEQ ID NOs:9-42, and CDR-H3 consisting of aminoacid residue 78 to at least amino acid residue 96 of SEQ ID NOs:9-42,wherein said Fab fragment is capable of binding IL-17A/F. In anotheraspect, the antibody fragment or single-chain antibody comprises a Fabfragment selected from the group consisting of the amino acid sequenceshown in FIG. 6 as SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42, wherein said Fab fragment further comprises at least one ofheavy chain variable region containing CDR-H1 consisting of amino acidresidues 7 to 16 of SEQ ID NOs:9-42, CDR-H2 consisting of amino acidresidues 30 to 46 of SEQ ID NOs:9-42, or CDR-H3 consisting of amino acidresidue 78 to at least amino acid residue 96 of SEQ ID NOs:9-42, whereinsaid Fab fragment is capable of binding IL-17A/F. In another aspect,said CDR-H1 region of SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ IDNO:42 comprises at least amino acid residues 7-10 corresponding to theamino sequence GFTI (designated herein as SEQ ID NO:77), wherein saidSEQ ID NO:77 is capable of binding IL-17A/F. In another aspect, saidCDR-H2 region of SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42comprises at least amino acid residues 41-46 corresponding to amino acidsequence YADSVK (designated herein as SEQ ID NO:78), wherein said SEQ IDNO:78 is capable of binding IL-17A/F.

In still another embodiment, the invention concerns an isolated nucleicacid molecule selected from the group consisting of the nucleotidesequence of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76,wherein said nucleic acid molecule encodes the Fab fragment shown as SEQID NO:9, SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42, wherein said Fabfragment is capable of binding to IL-17A/F.

In a another aspect, the invention provides an isolated Fab fragmentcapable of binding IL-17A/F encoded by a nucleotide sequence thatencodes such an amino acid sequence as hereinbefore described. Processesfor producing the same are also herein described, wherein thoseprocesses comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of said Fab fragment and recoveringsaid Fab fragment from the cell culture.

In yet another embodiment, the present invention provides a compositioncomprising an anti-IL-17A/F antibody in admixture with apharmaceutically acceptable carrier. In one aspect, the compositioncomprises a therapeutically effective amount of the antibody.Preferably, the composition is sterile. The composition may beadministered in the form of a liquid pharmaceutical formulation, whichmay be preserved to achieve extended storage stability. Alternatively,the antibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

(a) a composition of matter comprising an IL-17A/F polypeptide oragonist, antagonist, or an antibody that specifically binds to saidpolypeptide thereof;(b) a container containing said composition; and(c) a label affixed to said container, or a package insert included insaid container referring to the use of said IL-17A/F polypeptide oragonist or antagonist thereof in the treatment of an immune relateddisease. The composition may comprise a therapeutically effective amountof the IL-17A/F polypeptide or the agonist or antagonist thereof.

In yet another embodiment, the present invention concerns a method ofdiagnosing an immune related disease in a mammal, comprising detectingthe level of expression of a gene encoding an IL-17A/F polypeptide (a)in a test sample of tissue cells obtained from the mammal, and (b) in acontrol sample of known normal tissue cells of the same cell type,wherein a higher or lower expression level in the test sample ascompared to the control sample indicates the presence of immune relateddisease in the mammal from which the test tissue cells were obtained.

In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting ananti-IL-17A/F antibody with a test sample of tissue cells obtained fromthe mammal, and (b) detecting the formation of a complex between theantibody and an IL-17A/F polypeptide, in the test sample; wherein theformation of said complex is indicative of the presence or absence ofsaid disease. The detection may be qualitative or quantitative, and maybe performed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger quantity of complexes formed in the test sample indicates thepresence or absence of an immune disease in the mammal from which thetest tissue cells were obtained. The antibody preferably carries adetectable label. Complex formation can be monitored, for example, bylight microscopy, flow cytometry, fluorimetry, or other techniques knownin the art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality of the immune system.

In another embodiment, the invention provides a method for determiningthe presence of an IL-17A/F polypeptide in a sample comprising exposinga test sample of cells suspected of containing the IL-17A/F polypeptideto an anti-IL-17A/F antibody and determining the binding of saidantibody to said cell sample. In a specific aspect, the sample comprisesa cell suspected of containing the IL-17A/F polypeptide and the antibodybinds to the cell. The antibody is preferably detectably labeled and/orbound to a solid support.

In another embodiment, the present invention concerns an immune-relateddisease diagnostic kit, comprising an anti-IL-17A/F antibody and acarrier in suitable packaging. The kit preferably contains instructionsfor using the antibody to detect the presence of the IL-17A/Fpolypeptide. Preferably the carrier is pharmaceutically acceptable.

In another embodiment, the present invention concerns a diagnostic kit,containing an anti-IL-17A/F antibody in suitable packaging. The kitpreferably contains instructions for using the antibody to detect theIL-17A/F polypeptide.

In another embodiment, the invention provides a method of diagnosing animmune-related disease in a mammal which comprises detecting thepresence or absence or an IL-17A/F polypeptide in a test sample oftissue cells obtained from said mammal, wherein the presence or absenceof the IL-17A/F polypeptide in said test sample is indicative of thepresence of an immune-related disease in said mammal.

In another embodiment, the present invention concerns a method foridentifying an agonist of an IL-17A/F polypeptide comprising:

(a) contacting cells and a test compound to be screened under conditionssuitable for the induction of a cellular response normally induced by anIL-17A/F polypeptide; and (b) determining the induction of said cellularresponse to determine if the test compound is an effective agonist,wherein the induction of said cellular response is indicative of saidtest compound being an effective agonist.

In another embodiment, the invention concerns a method for identifying acompound capable of inhibiting the activity of an IL-17A/F polypeptidecomprising contacting a candidate compound with an IL-17A/F polypeptideunder conditions and for a time sufficient to allow these two componentsto interact and determining whether the activity of the IL-17A/Fpolypeptide is inhibited. In a specific aspect, either the candidatecompound or the IL-17A/F polypeptide is immobilized on a solid support.In another aspect, the non-immobilized component carries a detectablelabel. In a preferred aspect, this method comprises the steps of:

(a) contacting cells and a test compound to be screened in the presenceof an IL-17A/F polypeptide under conditions suitable for the inductionof a cellular response normally induced by an IL-17A/F polypeptide; and(b) determining the induction of said cellular response to determine ifthe test compound is an effective antagonist.

In another embodiment, the invention provides a method for identifying acompound that inhibits the expression of an IL-17A/F polypeptide incells that normally express the polypeptide, wherein the methodcomprises contacting the cells with a test compound and determiningwhether the expression of the IL-17A/F polypeptide is inhibited. In apreferred aspect, this method comprises the steps of:

(a) contacting cells and a test compound to be screened under conditionssuitable for allowing expression of the IL-17A/F polypeptide; and (b)determining the inhibition of expression of said polypeptide.

In yet another embodiment, the present invention concerns a method fortreating an immune-related disorder in a mammal that suffers therefromcomprising administering to the mammal a nucleic acid molecule thatcodes for either (a) an IL-17A/F polypeptide, (b) an agonist of anIL-17A/F polypeptide or (c) an antagonist of an IL-17A/F polypeptide,wherein said agonist or antagonist may be an anti-IL-17A/F antibody. Ina preferred embodiment, the mammal is human. In another preferredembodiment, the nucleic acid is administered via ex vivo gene therapy.In a further preferred embodiment, the nucleic acid is comprised withina vector, more preferably an adenoviral, adeno-associated viral,lentiviral or retroviral vector.

In yet another aspect, the invention provides a recombinant viralparticle comprising a viral vector consisting essentially of a promoter,nucleic acid encoding (a) an IL-17A/F polypeptide, (b) an agonistpolypeptide of an IL-17A/F polypeptide, or (c) an antagonist polypeptideof an IL-17A/F polypeptide, and a signal sequence for cellular secretionof the polypeptide, wherein the viral vector is in association withviral structural proteins. Preferably, the signal sequence is from amammal, such as from a native IL-17A/F polypeptide.

In a still further embodiment, the invention concerns an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) anIL-17A/F polypeptide, (b) an agonist polypeptide of an IL-17A/Fpolypeptide or (c) an antagonist polypeptide of an IL-17A/F polypeptide,and a signal sequence for cellular secretion of the polypeptide, whereinsaid producer cell packages the retroviral vector in association withthe structural proteins to produce recombinant retroviral particles.

In a still further embodiment, the invention provides a method forenhancing the infiltration of inflammatory cells from the vasculatureinto a tissue of a mammal comprising administering to said mammal (a) anIL-17A/F polypeptide or (b) an agonist of an IL-17A/F polypeptide,wherein the infiltration of inflammatory cells from the vasculature inthe mammal is enhanced.

In a still further embodiment, the invention provides a method fordecreasing the infiltration of inflammatory cells from the vasculatureinto a tissue of a mammal comprising administering to said mammal (a) anIL-17A/F polypeptide or (b) an antagonist of an IL-17A/F polypeptide,wherein the infiltration of inflammatory cells from the vasculature inthe mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17A/F polypeptide or (b) anagonist of an IL-17A/F polypeptide, wherein the activity ofT-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17A/F polypeptide or (b) anantagonist of an IL-17A/F polypeptide, wherein the activity ofT-lymphocytes in the mammal is decreased.

In a still further embodiment, the invention provides a method ofincreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17A/F polypeptide or (b) anagonist of an IL-17A/F polypeptide, wherein the proliferation ofT-lymphocytes in the mammal is increased.

In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) an IL-17A/F polypeptide or (b) anantagonist of an IL-17A/F polypeptide, wherein the proliferation ofT-lymphocytes in the mammal is decreased.

In still a further embodiment, the invention concerns the use of anIL-17A/F polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-IL-17A/F antibody, for thepreparation of a medicament useful in the treatment of a condition whichis responsive to the IL-17A/F polypeptide or an agonist or antagonistthereof (e.g., anti-IL-17A/F). In a particular aspect, the inventionconcerns the use of an IL-17A/F polypeptide, or an agonist or antagonistthereof in a method for treating a degenerative cartilaginous disorder.

In still a further embodiment, the invention relates to a method oftreating a degenerative cartilaginous disorder in a mammal comprisingadministering a therapeutically effective amount of an IL-17A/Fpolypeptide, agonist, or antagonist thereof, to said mammal sufferingfrom said disorder.

In still a further embodiment, the invention relates to a kit comprisinga composition comprising an IL-17A/F polypeptide, or an agonist orantagonist thereof, in admixture with a pharmaceutically acceptablecarrier; a container containing said composition; and a label affixed tosaid container, referring to the use of said composition, in thetreatment of a degenerative cartilaginous disorder.

B. Additional Embodiments

In other embodiments of the present invention, the invention provides anisolated nucleic acid molecule comprising a nucleotide sequence thatencodes an IL-17A/F polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding an IL-17A/F polypeptide having a full-length aminoacid sequence as disclosed herein, an amino acid sequence lacking thesignal peptide as disclosed herein, or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein, or(b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length IL-17A/F polypeptide cDNA as disclosed herein, the codingsequence of an IL-17A/F polypeptide lacking the signal peptide asdisclosed herein, or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

Another embodiment is directed to fragments of an IL-17A/F polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of an IL-17A/Fpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-IL-17A/F antibody or as antisenseoligonucleotide probes. Such nucleic acid fragments are usually at leastabout 20 nucleotides in length, alternatively at least about 30nucleotides in length, alternatively at least about 40 nucleotides inlength, alternatively at least about 50 nucleotides in length,alternatively at least about 60 nucleotides in length, alternatively atleast about 70 nucleotides in length, alternatively at least about 80nucleotides in length, alternatively at least about 90 nucleotides inlength, alternatively at least about 100 nucleotides in length,alternatively at least about 110 nucleotides in length, alternatively atleast about 120 nucleotides in length, alternatively at least about 130nucleotides in length, alternatively at least about 140 nucleotides inlength, alternatively at least about 150 nucleotides in length,alternatively at least about 160 nucleotides in length, alternatively atleast about 170 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 190 nucleotides inlength, alternatively at least about 200 nucleotides in length,alternatively at least about 250 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 350nucleotides in length, alternatively at least about 400 nucleotides inlength, alternatively at least about 450 nucleotides in length,alternatively at least about 500 nucleotides in length, alternatively atleast about 600 nucleotides in length, alternatively at least about 700nucleotides in length, alternatively at least about 800 nucleotides inlength, alternatively at least about 900 nucleotides in length andalternatively at least about 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length. It is noted that novelfragments of an IL-17A/F polypeptide-encoding nucleotide sequence may bedetermined in a routine manner by aligning the IL-17A/Fpolypeptide-encoding nucleotide sequence with other known nucleotidesequences using any of a number of well known sequence alignmentprograms and determining which polypeptide-encoding nucleotide sequencefragment(s) are novel. All of such polypeptide-encoding nucleotidesequences are contemplated herein. Also contemplated are the polypeptidefragments encoded by these nucleotide molecule fragments, preferablythose IL-17A/F polypeptide fragments that comprise a binding site for ananti-IL-17A/F antibody.

In another embodiment, the invention provides an isolated IL-17A/Fpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a certain aspect, the invention concerns an isolated IL-17A/Fpolypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anIL-17A/F polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein.

In a further aspect, the invention concerns an isolated IL-17A/Fpolypeptide comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated IL-17A/Fpolypeptide comprising an amino acid sequence scoring at least about 80%positives, alternatively at least about 81% positives, alternatively atleast about 82% positives, alternatively at least about 83% positives,alternatively at least about 84% positives, alternatively at least about85% positives, alternatively at least about 86% positives, alternativelyat least about 87% positives, alternatively at least about 88%positives, alternatively at least about 89% positives, alternatively atleast about 90% positives, alternatively at least about 91% positives,alternatively at least about 92% positives, alternatively at least about93% positives, alternatively at least about 94% positives, alternativelyat least about 95% positives, alternatively at least about 96%positives, alternatively at least about 97% positives, alternatively atleast about 98% positives and alternatively at least about 99% positiveswhen compared with the amino acid sequence of an IL-17A/F polypeptidehaving a full-length amino acid sequence as disclosed herein, an aminoacid sequence lacking the signal peptide as disclosed herein, or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein.

In a specific aspect, the invention provides an isolated IL-17A/Fpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as hereinbefore described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the IL-17A/F polypeptide and recovering the IL-17A/Fpolypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native IL-17A/F polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-IL-17A/Fantibody or a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to an IL-17A/F polypeptide which comprisecontacting the IL-17A/F polypeptide with a candidate molecule andmonitoring a biological activity mediated by said IL-17A/F polypeptide.Preferably, the IL-17A/F polypeptide is a native IL-17A/F polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising an IL-17A/F polypeptide, or an agonist or antagonistof an IL-17A/F polypeptide as herein described, or an anti-IL-17A/Fantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of anIL-17A/F polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-IL-17A/F antibody, for thepreparation of a medicament useful in the treatment of a condition whichis responsive to the IL-17A/F polypeptide, an agonist or antagonistthereof or an anti-IL-17A/F antibody.

In additional embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, yeast, orBaculovirus-infected insect cells. An process for producing any of theherein described polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of thedesired polypeptide and recovering the desired polypeptide from the cellculture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In yet another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of expressing and isolating a novel humancytokine designated IL-17A/F. Human 293 kidney cells were transfectedwith cDNA expression vectors encoding human IL-17 and IL-17F alone or incombination as indicated in FIG. 1A and FIG. 1B. Conditioned media fromtransfected cells was immunoprecipitated (IP) utilizing antibodies thatare able to recognize IL-17 (lanes 1-5), or IL-17F (lanes 6-10) asindicated in FIG. 1A and FIG. 1B. Western Blot analysis is showndemonstrating the presence of a dimeric IL-17A/F complex in lane 8 ofFIG. 1A and in lane 3 of FIG. 1B. The dimeric IL-17A/F complex isconsistent in size with a covalent heterodimeric species comprised ofone polypeptide chain of IL-17 and one polypeptide chain of IL-17F.

FIG. 2 shows the purification of recombinant IL-17A/F. FIG. 2A shows theresults of silver stained SDS-PAGE of protein fractions from initialfractionation of IL-17A/F on an S-Sepharose column. Fractions 31 and 32contains a protein with an apparent molecular mass of approximately 33kD consistent with IL-17A/F. FIG. 2B shows the results of furtherpurification of IL-17A/F using Vydac C4 column chromatography. Shown isthe chromatograph of eluted proteins measured at 214 nm and 280 nm. FIG.2C demonstrates that purified IL-17A/F protein fractions from the VydacC4 purification column induce IL-8 production in TK-10 cells.

FIG. 3 shows the results of amino acid sequence analysis of IL-17A/F.FIG. 3A shows the non-reducing SDS-PAGE analysis of purified IL-17A/F.Resolved protein was transferred to a PVDF membrane and stained withCoomassie blue protein stain. The positions of molecular weight markersare indicated on the right side. FIG. 3B shows the results of N-terminalsequence analysis of isolated IL-17A/F (amino acid residues detectedfrom an N-terminal sequence analysis of the band shown in FIG. 3A). Thesequence analysis reveals two N-terminal sequences (Sequence 1 isdesignated SEQ ID NO:1 and Sequence 2 is designated SEQ ID NO:2,respectively). FIG. 3C shows the amino acid sequence of human IL-17(shown in both FIG. 3C and FIG. 8, designated SEQ ID NO:3) and the aminoacid sequence of human IL-17F (shown both in FIG. 3C and FIG. 10,designated SEQ ID NO:4). The signal sequences of IL-17 and IL-17F areunderlined. The sequences that have identity to the two N-terminalpeptide sequences (SEQ ID NO: 1 and SEQ ID NO:2) present in IL-17A/F arehighlighted in bold for the shown IL-17 and IL-17F polypeptidesequences.

FIG. 4 shows mass spectrometry analysis of IL-17A/F. FIG. 4A is aschematic showing the amino acid sequence with its interchain andintrachain disulfide bonds of mature IL-17A/F heterodimer (SEQ IDNO:77). The cysteines involved in disulfide linkages are indicated bybullet, (*), and residue number. The disulfide bonds are indicated byblack lines connecting the bonded cysteines. Those disulfide bonds thatform interchain disulfide linkages are highlighted by bold black lines.FIG. 4B shows the schematic of IL-17A/F peptide fragments #1 and #2containing disulfide bonds between the IL-17 chain and the IL-17F chainthat would be anticipated to be produced by digestion of IL-17A/F withtrypsin [IL-17A/F disulfide bond fragment #1 is designated SEQ ID NO:7;IL-17A/F disulfide bond fragment #2 is designated SEQ ID NO:8,respectively]. The amino acids contained within these fragments areindicated and numbered relative to the initiating methionine of eachchain. Also indicated is the calculated approximate molecular mass ofthese fragments that would be expected to be observed by massspectrometry. FIG. 4C shows the matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI-TOF)peptide map of IL-17A/F. The resulting peptide map contains peaks with[M+H]+=2420.12 Da and 3410.60 Da, consistent with the disulfide linkedpeptides. FIG. 4D demonstrates further characterization of non-reducedsamples of IL-17A/F by liquid-chromatography electrospray ionization iontrap mass spectrometry (LC-ESI-MS). The ion chromatograms represent(from top to bottom) the total ion chromatogram, reconstructed ionchromatogram (RIC) of IL-17A/F disulfide bond fragment #2 [M+2H]2+, andIL-17A/F disulfide bond fragment #1 [M+2H]3+. Peaks consistent with bothheterodimers were observed whereas no peaks above background chemicalnoise were observed at the anticipated masses for homodimeric peptides.

FIG. 5A shows the dose response curves comparing the proinflammatoryresponse induced by IL-17A/F, IL-17 and IL-17F. IL-17A/F, IL-17 andIL-17F were incubated with TK-10 cells at the indicated concentrationsfor 24 hours. IL-17A/F was shown to have potent IL-8 inducing activitywith substantial activity seen at sub-nM concentrations. FIG. 5B showsthe dose response curves comparing IL-6 induction by IL-17A/F, IL-17 andIL-17F. IL-17A/F, IL-17 and IL-17F were incubated with TK-10 cells atthe indicated concentrations for 24 hours. TK-10 conditioned media wascollected and analyzed by IL-6 ELISA.

FIG. 6 shows the amino acid sequence of the region of the heavy chainvariable region containing CDR H1-H3 from Fab that bind IL-17A/F. Shownis an alignment of a region of the predicted amino acid sequence ofthirty four (34) clones (SEQ ID NO:9 to SEQ ID NO:42, respectively) thatencode distinct antibody heavy chain sequences that are able to bind toIL-17A/F. The three heavy chain CDR regions (CDR-H1, CDR-H2, CDR-H3) areshaded.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:5) of a native sequenceIL-17 cDNA.

FIG. 8 shows the amino acid sequence (SEQ ID NO:3) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:6) of a native sequenceIL-17F cDNA.

FIG. 10 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:6 shown in FIG. 9.

FIG. 11 shows IL-17A/F ELISA measurements of IL-17A/F produced fromanti-CD3/anti-CD28 activated human T-cells.

FIG. 12 shows the specificity of the IL-17A/F ELISA wherein threefractions #31-#33 assayed in parallel were shown to contain nearlyequivalent quantities of IL-17A/F (IL-17A and IL-17F were used ascontrols).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

A “native sequence IL-17A/F polypeptide” comprises a polypeptide havingthe same amino acid sequence as the corresponding IL-17A/F polypeptidederived from nature. Such native sequence IL-17A/F polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence IL-17A/F polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific IL-17A/F polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence IL-17A/F polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acid sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the IL-17A/F polypeptidesdisclosed in the accompanying figures are shown to begin with methionineresidues designated herein as amino acid position 1 in the figures, itis conceivable and possible that other methionine residues locatedeither upstream or downstream from the amino acid position 1 in thefigures may be employed as the starting amino acid residue for theIL-17A/F polypeptides.

The approximate location of the “signal peptides” of the variousIL-17A/F polypeptides disclosed herein are shown in the presentspecification and/or the accompanying figures. It is noted, however,that the C-terminal boundary of a signal peptide may vary, but mostlikely by no more than about 5 amino acids on either side of the signalpeptide C-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng., 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res., 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“IL-17A/F polypeptide variant” means an active IL-17A/F polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence IL-17A/F polypeptidesequence as disclosed herein, an IL-17A/F polypeptide sequence lackingthe signal peptide as disclosed herein, or any other fragment of afull-length IL-17A/F polypeptide sequence as disclosed herein. SuchIL-17A/F polypeptide variants include, for instance, IL-17A/Fpolypeptides wherein one or more amino acid residues are added, ordeleted, at the − or C-terminus of the full-length native amino acidsequence. Ordinarily, an IL-17A/F polypeptide variant will have at leastabout 80% amino acid sequence identity, alternatively at least about 81%amino acid sequence identity, alternatively at least about 82% aminoacid sequence identity, alternatively at least about 83% amino acidsequence identity, alternatively at least about 84% amino acid sequenceidentity, alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence IL-17A/F polypeptide sequence as disclosedherein, an IL-17A/F polypeptide sequence lacking the signal peptide asdisclosed herein, or any other specifically defined fragment of afull-length IL-17A/F polypeptide sequence as disclosed herein.Ordinarily, IL-17A/F variant polypeptides are at least about 10 aminoacids in length, alternatively at least about 20 amino acids in length,alternatively at least about 30 amino acids in length, alternatively atleast about 40 amino acids in length, alternatively at least about 50amino acids in length, alternatively at least about 60 amino acids inlength, alternatively at least about 70 amino acids in length,alternatively at least about 80 amino acids in length, alternatively atleast about 90 amino acids in length, alternatively at least about 100amino acids in length, alternatively at least about 150 amino acids inlength, alternatively at least about 200 amino acids in length,alternatively at least about 300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the IL-17A/Fpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific IL-17AF polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence of ahypothetical polypeptide of interest, “Comparison Protein” representsthe amino acid sequence of a polypeptide against which the polypeptideof interest is being compared, and “X, “Y” and “Z” each representdifferent hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of thepolypeptide of interest having a sequence derived from the nativepolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the polypeptide of interest is being comparedwhich may be an IL-17A/F variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of thepolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of the “ComparisonProtein” of interest and the amino acid sequence B is the amino acidsequence of the polypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“IL-17A/F variant polynucleotide” or “IL-17A/F variant nucleic acidsequence” means a nucleic acid molecule which encodes an active IL-17AFpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence IL-17A/F polypeptide sequence as disclosedherein, a full-length native sequence IL-17A/F polypeptide sequencelacking the signal peptide as disclosed herein, or any other fragment ofa full-length IL-17AF polypeptide sequence as disclosed herein.Ordinarily, an IL-17A/F variant polynucleotide will have at least about80% nucleic acid sequence identity, alternatively at least about 81%nucleic acid sequence identity, alternatively at least about 82% nucleicacid sequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence IL-17A/F polypeptide sequence as disclosed herein, afull-length native sequence IL-17A/F polypeptide sequence lacking thesignal peptide as disclosed herein, or any other fragment of afull-length IL-17A/F polypeptide sequence as disclosed herein. Variantsdo not encompass the native nucleotide sequence.

Ordinarily, IL-17A/F variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toIL-17A/F-encoding nucleic acid sequences identified herein is defined asthe percentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the IL-17A/F nucleic acid sequence of interest,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “IL-17A/F-DNA”,wherein “IL-17A/F-DNA” represents a hypothetical IL-17A/F-encodingnucleic acid sequence of interest, “Comparison DNA” represents thenucleotide sequence of a nucleic acid molecule against which the“IL-17A/F-DNA” nucleic acid molecule of interest is being compared, and“N”, “L” and “V” each represent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of theIL-17A/F polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence IL-17A/F polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the IL-17AF polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant IL-17A/Fpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the IL-17AF polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the IL-17A/F polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, IL-17A/F variant polynucleotides are nucleic acidmolecules that encode an active IL-17A/F polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length IL-17A/Fpolypeptide as disclosed herein. IL-17A/F variant polypeptides may bethose that are encoded by an IL-17A/F variant polynucleotide.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (2) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes polypeptide in situ within recombinantcells, since at least one component of the IL-17A/F polypeptide naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” IL-17A/F polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include an promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; an promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising an IL-17A/F polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native IL-17A/F polypeptide disclosed herein.In a similar manner, the term “agonist” is used in the broadest senseand includes any molecule that mimics a biological activity of a nativeIL-17A/F polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativeIL-17A/F polypeptides, peptides, antisense oligonucleotides, smallorganic molecules, etc. Methods for identifying agonists or antagonistsof an IL-17A/F polypeptide may comprise contacting an IL-17A/Fpolypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the IL-17A/F polypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-IL-17A/F monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies),anti-IL-17A/F antibody compositions with polyepitopic specificity,polyclonal antibodies, single chain anti-IL-17A/F antibodies, andfragments of anti-IL-17A/F antibodies (see below) as long as theyexhibit the desired biological or immunological activity. The term“immunoglobulin” (Ig) is used interchangeable with antibody herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight. (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(II) domains for μ and ε isotypes. Each V_(L) chainhas at the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H) 1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the V_(H); Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl, Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H) 1,C_(H) 2 and C_(H) 3. The constant domains may be native sequenceconstant domains (e.g. human native sequence constant domains) or aminoacid sequence variant thereof. Preferably, the intact antibody has oneor more effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H) 1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H) 1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fe region, which region is also the partrecognized by Fec receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinterchain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody,” e.g., a mammalian anti-human lgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10^(−s) and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

An “IL-17A/F binding oligopeptide” is an oligopeptide that binds,preferably specifically, to an IL-17A/F polypeptide as described herein.IL-17A/F binding oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. IL-17A/F binding oligopeptides are usually atleast about 5 amino acids in length, alternatively at least about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acids in length or more, wherein such oligopeptidesthat are capable of binding, preferably specifically, to an IL-17A/Fpolypeptide as described herein. IL-17A/F binding oligopeptides may beidentified without undue experimentation using well known techniques. Inthis regard, it is noted that techniques for screening oligopeptidelibraries for oligopeptides that are capable of specifically binding toa polypeptide target are well known in the art (see, e.g., U.S. Pat.Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484,5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564;Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysenet al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen etal., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci.USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991),J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

An “IL-17A/F binding organic molecule” is an organic molecule other thanan oligopeptide or antibody as defined herein that binds, preferablyspecifically, to an IL-17A/F polypeptide as described herein. IL-17A/Fbinding organic molecules may be identified and chemically synthesizedusing known methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). IL-17A/F binding organic molecules are usually less thanabout 2000 daltons in size, alternatively less than about 1500, 750,500, 250 or 200 daltons in size, wherein such organic molecules that arecapable of binding, preferably specifically, to an IL-17A/F polypeptideas described herein may be identified without undue experimentationusing well known techniques. In this regard, it is noted that techniquesfor screening organic molecule libraries for molecules that are capableof binding to a polypeptide target are well known in the art (see, e.g.,PCT Publication Nos. WO00/00823 and WO00/39585).

An antibody, oligopeptide or other organic molecule “which binds” anantigen of interest, e.g. a tumor-associated polypeptide antigen target,is one that binds the antigen with sufficient affinity such that theantibody, oligopeptide or other organic molecule is useful as adiagnostic and/or therapeutic agent in targeting a cell or tissueexpressing the antigen, and does not significantly cross-react withother proteins. In such embodiments, the extent of binding of theantibody, oligopeptide or other organic molecule to a “non-target”protein will be less than about 10% of the binding of the antibody,oligopeptide or other organic molecule to its particular target proteinas determined by fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). With regard to the binding of anantibody, oligopeptide or other organic molecule to a target molecule,the term “specific binding” or “specifically binds to” or is “specificfor” a particular polypeptide or an epitope on a particular polypeptidetarget means binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. The term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetas used herein can be exhibited, for example, by a molecule having a Kdfor the target of at least about 10⁻⁴ M, alternatively at least about10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternatively at leastabout 10⁻⁷ M, alternatively at least about 10⁻⁸ M, alternatively atleast about 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, alternativelyat least about 10⁻¹¹ M, alternatively at least about 10⁻¹² M, orgreater. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

An antibody, oligopeptide or other organic molecule that “inhibits thegrowth of tumor cells expressing an “IL-17A/F polypeptide” or a “growthinhibitory” antibody, oligopeptide or other organic molecule is onewhich results in measurable growth inhibition of cancer cells expressingor overexpressing the appropriate IL-17A/F polypeptide. Preferred growthinhibitory anti-IL-17A/F antibodies, oligopeptides or organic moleculesinhibit growth of IL-17A/F-expressing tumor cells by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g., from about 50% to about 100%) as compared to theappropriate control, the control typically being tumor cells not treatedwith the antibody, oligopeptide or other organic molecule being tested.In one embodiment, growth inhibition can be measured at an antibodyconcentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cellculture, where the growth inhibition is determined 1-10 days afterexposure of the tumor cells to the antibody. Growth inhibition of tumorcells in vivo can be determined in various ways. The antibody is growthinhibitory in vivo if administration of the anti-IL-17A/F antibody atabout 1 μg/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

An antibody, oligopeptide or other organic molecule which “inducesapoptosis” is one which induces programmed cell death as determined bybinding of annexin V, fragmentation of DNA, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is usually one whichoverexpresses an IL-17A/F polypeptide. Preferably the cell is a tumorcell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung,kidney, colon, bladder cell. Various methods are available forevaluating the cellular events associated with apoptosis. For example,phosphatidyl serine (PS) translocation can be measured by annexinbinding; DNA fragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, theantibody, oligopeptide or other organic molecule which induces apoptosisis one which results in about 2 to 50 fold, preferably about 5 to 50fold, and most preferably about 10 to 50 fold, induction of annexinbinding relative to untreated cell in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C Ilq bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hcmatopoictic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci.U.S.A. 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fe regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Cl q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as an IL-17A/F polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “modulate” means to affect (e.g., either upregulate,downregulate or otherwise control) the level of a signaling pathway.Cellular processes under the control of signal transduction include, butare not limited to, transcription of specific genes, normal cellularfunctions, such as metabolism, proliferation, differentiation, adhesion,apoptosis and survival, as well as abnormal processes, such astransformation, blocking of differentiation and metastasis.

“Active” or “activity” for the purposes herein refers to form(s) of anIL-17A/F polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring IL-17A/F polypeptides, wherein“biological” activity refers to a biological function (either inhibitoryor stimulatory) caused by a native or naturally-occurring IL-17AFpolypeptide other than the ability to induce the production of anantibody against an antigenic epitope possessed by a native ornaturally-occurring IL-17A/F polypeptide and an “immunological” activityrefers to the ability to induce the production of an antibody against anantigenic epitope possessed by a native or naturally-occurring IL-17A/Fpolypeptide. One preferred biological activity includes inducingactivation of NF-κB and stimulation of the production of theproinflammatory chemokines IL-8 and IL-6. Another preferred biologicalactivity includes stimulation of peripheral blood mononuclear cells orCD4⁺ cells. Another preferred biological activity includes stimulationof the proliferation of T-lymphocytes. Another preferred biologicalactivity includes, for example, the release of TNF-α from THP1 cells.Another activity includes an enhancement of matrix synthesis inarticular cartilage. Alternatively, another activity includes promotingbreakdown of articular cartilage matrix as well as inhibiting matrixsynthesis. Another preferred biological activity includes modulating thelevel of the interleukin-17 signalling pathway during mild to severestages of inflammatory bowel disease or during stroke.

An “immunological” activity refers only to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally-occurring IL-17A/F polypeptide.

“Degenerative cartilagenous disorder” describes a host of disorders thatis characterized principally by the destruction of the cartilage matrix.Additional pathologies includes nitric oxide production, and elevatedproteoglycan breakdown. Exemplary disorders encompassed within thisdefinition, include, for example, arthritis (e.g., osteoarthritis,rheumatoid arthritis, psoriatic arthritis).

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, which can be treated according to theinvention include systemic lupus erythematosis, rheumatoid arthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmunc hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barrd syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease. Infectiousdiseases including viral diseases such as AIDS (HIV infection),hepatitis A, B, C, D, and E, herpes, etc., bacterial infections, fungalinfections, protozoal infections and parasitic infections. The term“effective amount” is a concentration or amount of an IL-17AFpolypeptide and/or agonist/antagonist which results in achieving aparticular stated purpose. An “effective amount” of an IL-17A/Fpolypeptide or agonist or antagonist thereof may be determinedempirically. Furthermore, a “therapeutically effective amount” is aconcentration or amount of an IL-17A/F polypeptide and/oragonist/antagonist which is effective for achieving a stated therapeuticeffect. This amount may also be determined empirically.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al., (WBSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin:proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or TI-17; a tumor necrosis factorsuch as TNF-α or TNF-β; and other polypeptide factors including leukemiainhibitory factor (LIF) and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

TABLE 1 /* * * C-C increased from 12 to 15 * Z is average of EQ * B isaverage of ND * match with stop is _M; stop-stop = 0; J (joker) match =0 */ #define _M −8 /* value of a match with a stop */ int _day[26][26] ={ /*   A B C D E F G H I J K L M N O P Q R S T U V W X Y Z */ /* A */ {2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1, 1, 0, 0,−6,0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2, 2,_M,−1, 1,0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */ {−2,−4,15,−5,−5,−4,−3,−3,−2,0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8, 0, 0,−5}, /* D */ { 0, 3,−5,4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /*E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0, 0,−3,−2, 1,_M,−1, 2,−1, 0, 0,0,−2,−7, 0,−4, 3}, /* F */ {−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2,0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0, 0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5,5,−2,−3, 0,−2,−4,−3, 0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */{−1, 1,−3, 1, 1,−2,−2, 6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3,0, 0, 2}, /* I */ {−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2,2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5, 0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */{−1, 0,−5, 0, 0,−5,−2, 0,−2, 0, 5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3,0,−4, 0}, /* L */ {−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6,4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2, 0,−1,−2}, /* M */ {−1,−2,−5,−3,−2,0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1, 0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */{ 0, 2,−4, 2, 1,−4, 0, 2,−2, 0, 1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4,0,−2, 1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */ { 0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };/*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* maxjumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gapslarger than this */ #define JMPS 1024 /* max jmps in an path */ #defineMX 4 /* save if there's at least MX−1 bases since last jmp */ #defineDMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty formismatched bases */ #define DINS0 8 /* penalty for a gap */ #defineDINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */#define PINS1 4 /* penalty per residue */ struct jmp {  short n[MAXJMP];/* size of jmp (neg for dely) */  unsigned shor x[MAXJMP]; /* base no.of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 −1 */struct diag {  int score; /* score at last jmp */  long offset; /*offset of prev block */  short ijmp; /* current jmp index */  struct jmpjp; /* list of jmps */ }; struct path {  int spc; /* number of leadingspaces */  short n[JMPS]; /* size of jmp (gap) */  int x[JMPS]; /* locof jmp (last elem before gap) */ }; char *ofile; /* output file name */char *namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name forerr msgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* bestdiag: nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /*total gaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy;/* total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  *  where file1 and file2 are two dna or twoprotein sequences.  *  The sequences can be in upper- or lower-case anmay contain ambiguity  *  Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  *  Max file length is 65535 (limited by unsigned short x in thejmp struct)  *  A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  *  Output is in the file “align.out”  *  * Theprogram may create a tmp file in /tmp to hold info about traceback.  *Original version developed under BSD 4.3 on a vax 8650  */ #include“nw.h” #include “day.h” static _dbval[26] = { 1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = {  1, 2|(1<<(‘D’−‘A’))|(1<<(‘N’−‘A’)), 4, 8, 16, 32, 64, 128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14,  1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22,  1<<23, 1<<24,1<<25|(1<<(‘E’−‘A’))|<<(‘Q’−‘A’)) }; main(ac, av) main  int ac;  char*av[ ]; {  prog = av[0];  if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\”align.out\“\n”); exit(1);  }  namex[0] = av[1];  namex[1] = av[2]; seqx[0] = getseq(namex[0], &len0);  seqx[1] = getseq(namex[1], &len1); xbm = (dna)? _dbval : _pbval;  endgaps = 0; /* 1 to penalize endgaps */ ofile = “align.out”; /* output file */  nw( ); /* fill in the matrix,get the possible jmps */  readjmps( ); /* get the actual jmps */  print(); /* print stats, alignment */  cleanup(0); /* unlink any tmp files */} /* do the alignment, return best score: main( )  * dna: values inFitch and Smith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  *When scores are equal, we prefer mismatches to any gap, prefer  * a newgap to extending an ongoing gap, and prefer a gap in seqx  * to a gap inseq y.  */ nw( ) nw {  char *px, *py; /* seqs and ptrs */  int *ndely,*dely; /* keep track of dely */  int ndelx, delx; /* keep track of delx*/  int *tmp; /* for swapping row0, row1 */  int mis; /* score for eachtype */  int ins0, ins1; /* insertion penalties */  register id; /*diagonal index */  register ij; /* jmp index */  register *col0, *col1;/* score for curr, last row */  register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc(“to get diags”, len0+len1+1, sizeof(structdiag));  ndely = (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int *)g_calloc(“to get dely”, len1+1, sizeof(int));  col0 =(int *)g_calloc(“to get col0”, len1+1, sizeof(int));  col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int));  ins0 = (dna)? DINS0 :PINS0;  ins1 = (dna)? DINS1 : PINS1;  smax = −10000;  if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */  }  else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0;  /* fill in match matrix   */  for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy); if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx< len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax= col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void)free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print( ) -- only routinevisible outside this module  *  * static:  * getmat( ) -- trace backbest path, count matches: print( )  * pr_align( ) -- print alignment ofdescribed in array p[ ]: print( )  * dumpblock( ) -- dump a block oflines with numbers, stars: pr_align( )  * nums( ) -- put out a numberline: dumpblock( )  * putline( ) -- put out a line (name, [num], seq,[num]): dumpblock( )  * stars( ) - -put a line of stars: dumpblock( )  *stripname( ) -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC 3 #define P_LINE 256 /* maximum output line */#define P_SPC 3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print( ) print {  int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr,“%s: can't write%s\n”, prog, ofile); cleanup(1);  }  fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0);  fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1);  olen = 60;  lx = len0;  ly = len1; firstgap = lastgap = 0;  if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc;  }  else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc;  }  if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap;  }  else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap;  }  getmat(lx, ly, firstgap, lastgap);  pr_align( ); } /*  *trace back the best path, count matches  */ static getmat(lx, ly,firstgap, lastgap) getmat  int lx, ly; /* “core” (minus endgaps) */  intfirstgap, lastgap; /* leading trailing overlap */ {  int nm, i0, i1,siz0, siz1;  char outx[32];  double pct;  register n0, n1;  registerchar *p0, *p1;  /* get total matches, score   */  i0 = i1 = siz0 = siz1= 0;  p0 = seqx[0] + pp[1].spc;  p1 = seqx[1] + pp[0].spc;  n0 =pp[1].spc + 1;  n1 = pp[0].spc + 1;  nm = 0;  while ( *p0 && *p1 ) { if(siz0) { p1++; n1++; siz0−−; } else if (siz1) { p0++; n0++; siz1−−; }else { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0])siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++];p0++; p1++; }  }  /* pct homology:   * if penalizing endgaps, base isthe shorter seq   * else, knock off overhangs and take shorter core   */ if (endgaps) lx = (len0 < len1)? len0 : len1;  else lx = (lx < ly)? lx: ly;  pct = 100.*(double)nm/(double)lx;  fprintf(fx, “\n”); fprintf(fx, “<%d match%s in an overlap of %d: %.2f percentsimilarity\n”, nm, (nm == 1)? “” : “es”, lx, pct); fprintf(fx, “<gaps infirst sequence: %d”, gapx); ...getmat  if (gapx) { (void) sprintf(outx,“ (%d %s%s)”, ngapx, (dna)? “base”:“residue”, (ngapx == 1)? “”:“s”);fprintf(fx,“%s”, outx);  fprintf(fx, “, gaps in second sequence: %d”,gapy);  if (gapy) { (void) sprintf(outx, “ (%d %s%s)”, ngapy, (dna)?“base”:“residue”, (ngapy == 1)? “”:“s”); fprintf(fx,“%s”, outx);  }  if(dna) fprintf(fx, “\n<score: %d (match = %d, mismatch = %d, gap penalty= %d + %d per base)\n”, smax, DMAT, DMIS, DINS0, DINS1);  elsefprintf(fx, “\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %dper residue)\n”, smax, PINS0, PINS1);  if (endgaps) fprintf(fx,“<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n”,firstgap, (dna)? “base” : “residue”, (firstgap == 1)? “” : “s”, lastgap,(dna)? “base” : “residue”, (lastgap == 1)? “” : “s”);  else fprintf(fx,“<endgaps not penalized\n”); } static nm; /* matches in core -- forchecking */ static lmax; /* lengths of stripped file names */ staticij[2]; /* jmp index for a path */ static nc[2]; /* number at start ofcurrent line */ static ni[2]; /* current elem number -- for gapping */static siz[2]; static char *ps[2]; /* ptr to current element */ staticchar *po[2]; /* ptr to next output char slot */ static charout[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set bystars( ) */ /*  * print alignment of described in struct path pp[ ]  */static pr_align( ) pr_align {  int nn; /* char count */  int more; register i;  for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /*  *do we have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) { /* leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if(siz[i]) { /* in a gap */ *po[i]++ = ‘-’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; }  } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock {  register i;  for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock  (void) putc(‘\n’,fx);  for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ ||*(po[i]) != ‘ ’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i); if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1)nums(i); }  } } /*  * put out a number line: dumpblock( )  */ staticnums(ix) nums  int ix; /* index in out[ ] holding seq line */ {  charnline[P_LINE];  register i, j;  register char *pn, *px, *py;  for (pn =nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ‘ ’;  for (i = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py == ‘ ’ || *py == ‘-’) *pn = ‘ ’;else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i;for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘-’;} else *pn = ‘ ’; i++; }  }  *pn = ‘\0’;  nc[ix] = i;  for (pn = nline;*pn; pn++) (void) putc(*pn, fx);  (void) putc(‘\n’, fx); } /*  * put outa line (name, [num], seq, [num]): dumpblock( )  */ static putline(ix)putline  int ix; { ...putline  int i;  register char *px;  for (px =namex[ix], i = 0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void) putc(‘ ’, fx);  /* these count from1:   * ni[ ] is current element (from 1)   * nc[ ] is number at start ofcurrent line   */  for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx);  (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock( )  */ static stars( ) stars {  int i; register char *p0, *p1, cx, *px;  if (!*out[0] || (*out[0] == ‘ ’ &&*(po[0]) == ‘ ’) ||   !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’))return;  px = star;  for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’;  for (p0= out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx;  }  *px++ = ‘\n’;  *px = ‘\0’; } /*  * strip pathor prefix from pn, return len: pr_align( )  */ static stripname(pn)stripname  char *pn; /* file name (may be path) */ {  register char *px,*py;  py = 0;  for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1;  if(py) (void) strcpy(pn, py);  return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup  int i; {  if (fj)(void) unlink(jname);  exit(i); } /*  * read, return ptr to seq, setdna, len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq inupper or lower case  */ char * getseq(file, len) getseq  char *file; /*file name */  int *len; /* seq len */ {  char line[1024], *pseq; register char *px, *py;  int natgc, tlen;  FILE *fp;  if ((fp =fopen(file,“r”)) == 0) { fprintf(stderr,“%s: can't read %s\n”, prog,file); exit(1);  }  tlen = natgc = 0;  while (fgets(line, 1024, fp)) {if (*line == ‘;’ || *line == ‘<’ || *line == ‘>’) continue; for (px =line; *px != ‘\n’; px++) if (isupper(*px) || islower(*px)) tlen++;  } if ((pseq = malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s:malloc( ) failed to get %d bytes for %s\n”, prog, tlen+6, file);exit(1);  }  pseq[0] = pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq  py= pseq + 4;  *len = tlen;  rewind(fp);  while (fgets(line, 1024, fp)) {if (*line == ‘;’ || *line == ‘<’ || *line == ‘>’) continue; for (px =line; *px != ‘\n’; px++){ if (isupper(*px)) *py++ = *px; else if(islower(*px)) *py++ = toupper(*px); if (index(“ATGCU”,*(py−1)))natgc++; }  }  *py++ = ‘\0’;  *py = ‘\0’;  (void) fclose(fp);  dna =natgc > (tlen/3);  return(pseq+4); } char * g_calloc(msg, nx, sz)g_calloc  char *msg; /* program, calling routine */  int nx, sz; /*number and size of elements */ {  char *px, *calloc( );  if ((px =calloc((unsigned)nx, (unsigned)sz)) == 0) { if (*msg) { fprintf(stderr,“%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”, prog, msg, nx, sz);exit(1); }  }  return(px); } /*  * get final jmps from dx[ ] or tmpfile, set pp[ ], reset dmax: main( )  */ readjmps( ) readjmps {  int fd= −1;  int siz, i0, i1;  register i, j, xx;  if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open( ) %s\n”, prog, jname); cleanup(1); }  }  for (i = i0 =i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmps if(j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0);(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break;  }  /* reverse the orderof jmps   */  for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i;  }  for (j = 0, i1−−; j < i1; j++, i1−−) {i = pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i =pp[1].x[j]; pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i;  }  if (fd >= 0)(void) close(fd);  if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  * write a filled jmp struct offset of the prev one (if any):nw( )  */ writejmps(ix) writejmps  int ix; {  char *mktemp( );  if (!fj){ if (mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”,prog, jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); }  } (void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);  (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

TABLE 2 IL-17A/F XXXXXXXXXXXXXXX (Length = 15 amino acids) ProteinComparison XXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acidsequence identity = (the number of identically matching amino acidresidues between the two polypeptide sequences as determined by ALIGN-2)divided by (the total number of amino acid residues of the IL-17A/Fprotein) = 5 divided by 15 = 33.3%

TABLE 3 IL-17A/F Protein XXXXXXXXXX (Length = 10 amino acids) ComparisonProtein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the IL-17A/F protein) = 5divided by 10 = 50%

TABLE 4 IL-17A/F-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonDNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides) % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the IL-17A/F-DNA nucleic acid sequence) =6 divided by 14 = 42.9%

TABLE 5 IL-17A/F-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the IL-17A/F-DNA nucleic acid sequence) = 4divided by 12 = 33.3%

II. Compositions and Methods of the Invention

A. Full-Length IL-17A/F Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas IL-17A/F polypeptides. In particular, cDNAs encoding various IL-17A/Fpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below.

B. IL-17A/F Polypeptide Variants

In addition to the full-length native sequence IL-17A/F polypeptidesdescribed herein, it is contemplated that IL-17A/F variants can beprepared. IL-17A/F variants can be prepared by introducing appropriatenucleotide changes into the IL-17A/F DNA, and/or by synthesis of thedesired IL-17A/F polypeptide. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of theIL-17A/F, such as changing the number or position of glycosylation sitesor altering the membrane anchoring characteristics.

Variations in the native full-length sequence IL-17A/F or in variousdomains of the IL-17A/F described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the IL-17A/F that results in a change in theamino acid sequence of the IL-17A/F as compared with the native sequenceIL-17A/F. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe IL-17A/F. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the IL-17A/F withthat of homologous known protein molecules and minimizing the number ofamino acid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

IL-17A/F polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the IL-17A/F polypeptide.

IL-17A/F fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating IL-17A/F fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, IL-17A/F polypeptide fragments share atleast one biological and/or immunological activity with the nativeIL-17A/F polypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr tyr Thr (T)ser set Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of theIL-17A/F polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis (Wells et al., Gene, 34:315 [1985]),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 [1986]) or other known techniques can be performedon the cloned DNA to produce the IL-17A/F variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,Science, 244: 1081-1085 j1989J). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 [1976]). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of IL-17A/F

Covalent modifications of IL-17A/F are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of an IL-17A/F polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the IL-17A/F. Derivatization withbifunctional agents is useful, for instance, for crosslinking IL-17A/Fto a water-insoluble support matrix or surface for use in the method forpurifying anti-IL-17A/F antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of thea-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the IL-17A/F polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence IL-17A/F(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceIL-17A/F. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the IL-17A/F polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence IL-17A/F (forO-linked glycosylation sites). The IL-17A/F amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the IL-17A/F polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids.

Another means of increasing the number of carbohydrate moieties on theIL-17A/F polypeptide is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the IL-17A/F polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of IL-17A/F comprises linking theIL-17A/F polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The IL-17A/F of the present invention may also be modified in a way toform a chimeric molecule comprising IL-17A/F fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theIL-17A/F with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the IL-17A/F. The presenceof such epitope-tagged forms of the IL-17A/F can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the IL-17A/F to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3:547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an a-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the IL-17A/F with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fecregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of an IL-17A/F polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

In yet a further embodiment, the IL-17A/F polypeptides of the presentinvention may also be modified in a way to form a chimeric moleculecomprising an IL-17A/F polypeptide fused to a leucine zipper. Variousleucine zipper polypeptides have been described in the art. See, e.g.,Landschulz et al., Science 240:1759 (1988); WO 94/10308; Hoppe et al.,FEBS Letters, 344:1991 (1994); Maniatis et al., Nature, 341:24 (1989).It is believed that use of a leucine zipper fused to an IL-17A/Fpolypeptide may be desirable to assist in dimerizing or trimerizingsoluble IL-17A/F polypeptide in solution. Those skilled in the art willappreciate that the leucine zipper may be fused at either the N- orC-terminal end of the IL-17A/F molecule.

D. Preparation of IL-17A/F

The description below relates primarily to production of IL-17A/F byculturing cells transformed or transfected with a vector containingIL-17A/F nucleic acid. It is, of course, contemplated that alternativemethods, which are well known in the art, may be employed to prepareIL-17A/F. For instance, the IL-17A/F sequence, or portions thereof, maybe produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the IL-17A/F may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length IL-17A/F.

1. Isolation of DNA Encoding IL-17A/F

DNA encoding IL-17A/F may be obtained from a cDNA library prepared fromtissue believed to possess the IL-17A/F mRNA and to express it at adetectable level. Accordingly, human IL-17A/F DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The IL-17A/F-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to theIL-17A/F or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding IL-17A/F is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for IL-17A/F production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant IL-17A/F ductfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forIL-17A/F-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe [Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2May 19851; Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 [1991]) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. frugilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technologv, 8:135 [1990]), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene.26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA,81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated IL-17A/F arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9 orSpodoptera High 5 cells, as well as plant cells. Examples of usefulmammalian host cell lines include Chinese hamster ovary (CHO) and COScells. More specific examples include monkey kidney CV 1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]);mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]);human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selectionof the appropriate host cell is deemed to be within the skill in theart.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding IL-17A/F may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, an promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The IL-17A/F may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe IL-17A/F-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2p plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theIL-17A/F-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp 1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the IL-17A/F-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingIL-17A/F.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

IL-17A/F transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the IL-17A/F by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,a-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theIL-17A/F coding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding IL-17A/F.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of IL-17A/F in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et a.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceIL-17A/F polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused toIL-17A/F DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of IL-17A/F may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g., Triton-X 100) or by enzymaticcleavage. Cells employed in expression of IL-17A/F can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify IL-17A/F from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theIL-17A/F. Various methods of protein purification may be employed andsuch methods are known in the art and described for example inDeutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular IL-17A/Fproduced.

E. Uses for IL-17A/F

Nucleotide sequences (or their complement) encoding IL-17A/F havevarious applications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. IL-17A/F nucleic acid will also beuseful for the preparation of IL-17A/F polypeptides by the recombinanttechniques described herein.

The full-length native sequence IL-17A/F gene, or portions thereof, maybe used as hybridization probes for a cDNA library to isolate thefull-length IL-17A/F cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of IL-17A/F or IL-17A/F fromother species) which have a desired sequence identity to the nativeIL-17A/F sequence disclosed herein. Optionally, the length of the probeswill be about 20 to about 50 bases. The hybridization probes may bederived from at least partially novel regions of the full length nativenucleotide sequence wherein those regions may be determined withoutundue experimentation or from genomic sequences including promoters,enhancer elements and introns of native sequence IL-17A/F. By way ofexample, a screening method will comprise isolating the coding region ofthe IL-17A/F gene using the known DNA sequence to synthesize a selectedprobe of about 40 bases. Hybridization probes may be labeled by avariety of labels, including radionucleotides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the IL-17A/F gene of the present invention canbe used to screen libraries of human cDNA, genomic DNA or mRNA todetermine which members of such libraries the probe hybridizes to.Hybridization techniques are described in further detail in the Examplesbelow.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the IL-17A/F nucleic acids include antisenseor sense oligonucleotides comprising a singe-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target IL-17A/F mRNA(sense) or IL-17A/F DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of IL-17A/F DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, [1988])and van der Krol et al. (BioTechniques. 6:958, [1988]).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of IL-17A/Fproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related IL-17A/F codingsequences.

Nucleotide sequences encoding an IL-17A/F can also be used to constructhybridization probes for mapping the gene which encodes that IL-17A/Fand for the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for IL-17A/F encode a protein which binds toanother protein (example, where the protein is a receptor), the proteincan be used in assays to identify the other proteins or moleculesinvolved in the binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor protein can be used to isolate correlative ligand(s).Screening assays can be designed to find lead compounds that mimic thebiological activity of a native IL-17A/F or a receptor for IL-17A/F.Such screening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art.

Nucleic acids which encode IL-17A/F or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding IL-17A/F can be used to clone genomic DNAencoding IL-17A/F in accordance with established techniques and thegenomic sequences used to generate transgenic animals that contain cellswhich express DNA encoding IL-17A/F. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for IL-17A/F transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding IL-17A/F introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding IL-17A/F. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of IL-17A/F can be used to constructan IL-17A/F “knock out” animal which has a defective or altered geneencoding IL-17A/F as a result of homologous recombination between theendogenous gene encoding IL-17A/F and altered genomic DNA encodingIL-17A/F introduced into an embryonic stem cell of the animal. Forexample, cDNA encoding IL-17A/F can be used to clone genomic DNAencoding IL-17A/F in accordance with established techniques. A portionof the genomic DNA encoding IL-17A/F can be deleted or replaced withanother gene, such as a gene encoding a selectable marker which can beused to monitor integration. Typically, several kilobases of unalteredflanking DNA (both at the 5′ and 3′ ends) are included in the vector[see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description ofhomologous recombination vectors]. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected [see, e.g., Li et al., Cell, 69:915 (1992)]. The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse orrat) to form aggregation chimeras [see, e.g., Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term to create a “knock out” animal. Progenyharboring the homologously recombined DNA in their germ cells can beidentified by standard techniques and used to breed animals in which allcells of the animal contain the homologously recombined DNA. Knockoutanimals can be characterized for instance, for their ability to defendagainst certain pathological conditions and for their development ofpathological conditions due to absence of the IL-17A/F polypeptide.

Nucleic acid encoding the IL-17A/F polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA, 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology, 11: 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science, 256: 808-813(1992).

The IL-17A/F polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes and theisolated nucleic acid sequences may be used for recombinantly expressingthose markers.

The nucleic acid molecules encoding the IL-17A/F polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachIL-17A/F nucleic acid molecule of the present invention can be used as achromosome marker.

The IL-17A/F polypeptides and nucleic acid molecules of the presentinvention may also be used diagnostically for tissue typing, wherein theIL-17A/F polypeptides of the present invention may be differentiallyexpressed in one tissue as compared to another, preferably in a diseasedtissue as compared to a normal tissue of the same tissue type. IL-17A/Fnucleic acid molecules will find use for generating probes for PCR,Northern analysis, Southern analysis and Western analysis.

The IL-17A/F polypeptides described herein may also be employed astherapeutic agents. The IL-17A/F polypeptides of the present inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the IL-17A/F product hereof is combined inadmixture with a pharmaceutically acceptable carrier vehicle.Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.,injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of an IL-17A/F polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of an IL-17A/F polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of theIL-17A/F polypeptide, microencapsulation of the IL-17A/F polypeptide iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon- (rhIFN-), interleukin-2, and MN rgp120. Johnson etal., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223(1993); Hora et al., Bio/Technology, 8:755-758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the IL-17A/F polypeptide (agonists) or prevent theeffect of the IL-17A/F polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the IL-17A/F polypeptides encoded by the genesidentified herein, or otherwise interfere with the interaction of theencoded polypeptides with other cellular proteins. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with an IL-17A/F polypeptide encoded by a nucleicacid identified herein under conditions and for a time sufficient toallow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the IL-17A/F polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the IL-17A/F polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for theIL-17A/F polypeptide to be immobilized can be used to anchor it to asolid surface. The assay is performed by adding the non-immobilizedcomponent, which may be labeled by a detectable label, to theimmobilized component, e.g., the coated surface containing the anchoredcomponent. When the reaction is complete, the non-reacted components areremoved, e.g., by washing, and complexes anchored on the solid surfaceare detected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular IL-17A/F polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 [1989]);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 [1991]) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA,89:5789-5793 (1991). Many transcriptional activators, such as yeastGAL4, consist of two physically discrete modular domains, one acting asthe DNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for 3-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding anIL-17A/F polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed herein above. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the IL-17A/F polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the IL-17A/F polypeptide indicates that the compound isan antagonist to the IL-17A/F polypeptide. Alternatively, antagonistsmay be detected by combining the IL-17A/F polypeptide and a potentialantagonist with membrane-bound IL-17A/F polypeptide receptors orrecombinant receptors under appropriate conditions for a competitiveinhibition assay. The IL-17A/F polypeptide can be labeled, such as byradioactivity, such that the number of IL-17A/F polypeptide moleculesbound to the receptor can be used to determine the effectiveness of thepotential antagonist. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Coligan et al., Current Protocols inImmun., 1(2): Chapter 5 (1991). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto the IL-17A/F polypeptide and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the IL-17A/F polypeptide. Transfected cells thatare grown on glass slides are exposed to labeled IL-17A/F polypeptide.The IL-17A/F polypeptide can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase. Following fixation and incubation, the slides aresubjected to autoradiographic analysis. Positive pools are identifiedand sub-pools are prepared and re-transfected using an interactivesub-pooling and re-screening process, eventually yielding a single clonethat encodes the putative receptor.

As an alternative approach for receptor identification, labeled IL-17A/Fpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledIL-17A/F polypeptide in the presence of the candidate compound. Theability of the compound to enhance or block this interaction could thenbe measured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin withIL-17A/F polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of theIL-17A/F polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the IL-17A/F polypeptide.

Another potential IL-17A/F polypeptide antagonist is an antisense RNA orDNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature IL-17A/F polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the IL-17A/F polypeptide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the IL-17A/F polypeptide (antisense—Okano,Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression (CRC Press: Boca Raton, Fla., 1988). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the IL-17A/F polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the IL-17A/F polypeptide, thereby blocking the normalbiological activity of the 1-17A/F polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed herein above and/or by any other screeningtechniques well known for those skilled in the art.

Diagnostic and therapeutic uses of the herein disclosed molecules mayalso be based upon the positive functional assay hits disclosed anddescribed below

F. Tissue Distribution

The location of tissues expressing the IL-17A/F can be identified bydetermining mRNA expression in various human tissues. The location ofsuch genes provides information about which tissues are most likely tobe affected by the stimulating and inhibiting activities of the IL-17A/Fpolypeptides. The location of a gene in a specific tissue also providessample tissue for the activity blocking assays discussed below.

As noted before, gene expression in various tissues may be measured byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205[1980]), dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequences provided herein.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof an IL-17A/F polypeptide or against a synthetic peptide based on theDNA sequences encoding the IL-17A/F polypeptide or against an exogenoussequence fused to a DNA encoding an IL-17A/F polypeptide and encoding aspecific antibody epitope. General techniques for generating antibodies,and special protocols for Northern blotting and in situ hybridizationare provided below.

G. Antibody Binding Studies

The activity of the IL-17A/F polypeptides can be further verified byantibody binding studies, in which the ability of anti-IL-17A/Fantibodies to inhibit the effect of the IL-17A/F polypeptides,respectively, on tissue cells is tested. Exemplary antibodies includepolyclonal, monoclonal, humanized, bispecific, and heteroconjugateantibodies, the preparation of which will be described herein below.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme. For immunohistochemistry, the tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin, for example.

H. Cell-Based Assays

Cell-based assays and animal models for immune related diseases can beused to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

In a different approach, cells of a cell type known to be involved in aparticular immune related disease are transfected with the cDNAsdescribed herein, and the ability of these cDNAs to stimulate or inhibitimmune function is analyzed. Suitable cells can be transfected with thedesired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g.,Small et al., Mol. Cell. Biol., 5: 642-648 [1985]).

One suitable cell based assay is the mixed lymphocyte reaction (MLR).Current Protocols in Immunology, unit 3.12; edited by J E Coligan, A MKruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes ofHealth, Published by John Wiley & Sons, Inc. In this assay, the abilityof a test compound to stimulate or inhibit the proliferation ofactivated T cells is assayed. A suspension of responder T cells iscultured with allogeneic stimulator cells and the proliferation of Tcells is measured by uptake of tritiated thymidine. This assay is ageneral measure of T cell reactivity. Since the majority of T cellsrespond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, above, 3.15, 6.3.

A proliferative T cell response in an MLR assay may be due to directmitogenic properties of an assayed molecule or to external antigeninduced activation. Additional verification of the T cell stimulatoryactivity of the IL-17A/F polypeptides can be obtained by a costimulationassay. T cell activation requires an antigen specific signal mediatedthrough the T-cell receptor (TCR) and a costimulatory signal mediatedthrough a second ligand binding interaction, for example, the B7 (CD80,CD86)/CD28 binding interaction. CD28 crosslinking increases lymphokinesecretion by activated T cells. T cell activation has both negative andpositive controls through the binding of ligands which have a negativeor positive effect. CD28 and CTLA-4 are related glycoproteins in the Igsuperfamily which bind to B7. CD28 binding to B7 has a positivecostimulation effect of T cell activation; conversely, CTLA-4 binding toB7 has a negative T cell deactivating effect. Chambers, C. A. andAllison, J. P., Curr. Opin. Immunol., (1997) 9:396. Schwartz, R. H.,Cell (1992) 71:1065; Linsley, P. S. and Ledbetter, J. A., Annu. Rev.Immunol. (1993) 11:191; June, C. H. et al., Immunol. Today (1994)15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulation assay,the IL-17A/F polypeptides are assayed for T cell costimulatory orinhibitory activity.

IL-17A/F polypeptides, as well as other compounds of the invention,which are stimulators (costimulators) of T cell proliferation andagonists, e.g., agonist antibodies, thereto as determined by MLR andcostimulation assays, for example, are useful in treating immune relateddiseases characterized by poor, suboptimal or inadequate immunefunction. These diseases are treated by stimulating the proliferationand activation of T cells (and T cell mediated immunity) and enhancingthe immune response in a mammal through administration of a stimulatorycompound, such as the stimulating IL-17A/F polypeptides. The stimulatingpolypeptide may, for example, be an IL-17A/F polypeptide or an agonistantibody thereof.

Direct use of a stimulating compound as in the invention has beenvalidated in experiments with 4-IBB glycoprotein, a member of the tumornecrosis factor receptor family, which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol., 24:2219 (1994).

The use of an agonist stimulating compound has also been validatedexperimentally. Activation of 4-1BB by treatment with an agonistanti-4-1BB antibody enhances eradication of tumors. Hellstrom, I. andHellstrom, K. E., Crit. Rev. Immunol., 18:1 (1998). Immunoadjuvanttherapy for treatment of tumors, described in more detail below, isanother example of the use of the stimulating compounds of theinvention. An immune stimulating or enhancing effect can also beachieved by antagonizing or blocking the activity of an IL-17A/F whichhas been found to be inhibiting in the MLR assay. Negating theinhibitory activity of the compound produces a net stimulatory effect.Suitable antagonists/blocking compounds are antibodies or fragmentsthereof which recognize and bind to the inhibitory protein, therebyblocking the effective interaction of the protein with its receptor andinhibiting signaling through the receptor. This effect has beenvalidated in experiments using anti-CTLA-4 antibodies which enhance Tcell proliferation, presumably by removal of the inhibitory signalcaused by CTLA-4 binding. Walunas, T. L. et al., Immunity, 1:405 (1994).

Alternatively, an immune stimulating or enhancing effect can also beachieved by administration of an IL-17A/F polypeptide which has vascularpermeability enhancing properties. Enhanced vacuolar permeability wouldbe beneficial to disorders which can be attenuated by local infiltrationof immune cells (e.g., monocytes, eosinophils, PMNs) and inflammation.

On the other hand, IL-17A/F polypeptides, as well as other compounds ofthe invention, which are direct inhibitors of T cellproliferation/activation, lymphokine secretion, and/or vascularpermeability can be directly used to suppress the immune response. Thesecompounds are useful to reduce the degree of the immune response and totreat immune related diseases characterized by a hyperactive,superoptimal, or autoimmune response. This use of the compounds of theinvention has been validated by the experiments described above in whichCTLA-4 binding to receptor B7 deactivates T cells. The direct inhibitorycompounds of the invention function in an analogous manner. The use ofcompound which suppress vascular permeability would be expected toreduce inflammation. Such uses would be beneficial in treatingconditions associated with excessive inflammation.

Alternatively, compounds, e.g., antibodies, which bind to stimulatingIL-17A/F polypeptides and block the stimulating effect of thesemolecules produce a net inhibitory effect and can be used to suppressthe T cell mediated immune response by inhibiting T cellproliferation/activation and/or lymphokine secretion. Blocking thestimulating effect of the polypeptides suppresses the immune response ofthe mammal. This use has been validated in experiments using an anti-IL2antibody. In these experiments, the antibody binds to IL2 and blocksbinding of IL2 to its receptor thereby achieving a T cell inhibitoryeffect.

1. Animal Models

The results of the cell based in vitro assays can be further verifiedusing in vivo animal models and assays for T-cell function. A variety ofwell known animal models can be used to further understand the role ofthe genes identified herein in the development and pathogenesis ofimmune related disease, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them predictive of responses in humanpatients. Animal models of immune related diseases include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing cells into syngeneic mice usingstandard techniques, e.g., subcutaneous injection, tail vein injection,spleen implantation, intraperitoneal implantation, implantation underthe renal capsule, etc.

Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunolovgy, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction and a measureof their role in transplant rejection. The most common and acceptedmodels use murine tail-skin grafts. Repeated experiments have shown thatskin allograft rejection is mediated by T cells, helper T cells andkiller-effector T cells, and not antibodies. Auchincloss, H. Jr. andSachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., RavenPress, NY, 889-992 (1989). A suitable procedure is described in detailin Current Protocols in Immunology, above, unit 4.4. Other transplantrejection models which can be used to test the compounds of theinvention are the allogeneic heart transplant models described byTanabe, M. et al., Transplantation, 58:23 (1994) and Tinubu, S. A. etal., J. Immunol., 4330-4338 (1994).

Animal models for delayed type hypersensitivity provides an assay ofcell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell andmononuclear cell inflammation and subsequent demyelination of axons inthe central nervous system. EAE is generally considered to be a relevantanimal model for MS in humans. Bolton, C., Multiple Sclerosis, 1:143(1995). Both acute and relapsing-remitting models have been developed.The compounds of the invention can be tested for T cell stimulatory orinhibitory activity against immune mediated demyelinating disease usingthe protocol described in Current Protocols in Immunology, above, units15.1 and 15.2. See also the models for myelin disease in whicholigodendrocytes or Schwann cells are grafted into the central nervoussystem as described in Duncan, I. D. et al., Molec. Med. Today, 554-561(1997).

Contact hypersensitivity is a simple delayed type hypersensitivity invivo assay of cell mediated immune function. In this procedure,cutaneous exposure to exogenous haptens which gives rise to a delayedtype hypersensitivity reaction which is measured and quantitated.Contact sensitivity involves an initial sensitizing phase followed by anelicitation phase. The elicitation phase occurs when the T lymphocytesencounter an antigen to which they have had previous contact. Swellingand inflammation occur, making this an excellent model of human allergiccontact dermatitis. A suitable procedure is described in detail inCurrent Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc.,unit 4.2 (1994). I also Grabbe, S. and Schwarz, T, Immun. Today, 19(1):37-44 (1998).

An animal model for arthritis is collagen-induced arthritis. This modelshares clinical, histological and immunological characteristics of humanautoimmune rheumatoid arthritis and is an acceptable model for humanautoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CDI 8 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology, 88:569(1996).

A model of asthma has been described in which antigen-induced airwayhyper-reactivity, pulmonary eosinophilia and inflammation are induced bysensitizing an animal with ovalbumin and then challenging the animalwith the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al., Am. J.Respir. Cell Mol. Biol., 18:777 (1998) and the references cited therein.

Additionally, the compounds of the invention can be tested on animalmodels for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al., Nat.Med., 3:183 (1997), in which the mice demonstrate histopathologic skinlesions resembling psoriasis. Another suitable model is the humanskin/scid mouse chimera prepared as described by Nickoloff, B. J. etal., Am. J. Path., 146:580 (1995).

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g., baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82, 6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell, 56,313-321 [1989]); electroporation of embryos (Lo, Mol. Cel. Biol., 3,1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell.57, 717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA, 89, 6232-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to the IL-17A/F polypeptide,prepared as described above, are administered to the animal and theeffect on immune function is determined.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a polypeptide identified herein, as aresult of homologous recombination between the endogenous gene encodingthe polypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

J. Immunoadjuvant Therapy

In one embodiment, the immunostimulating compounds of the invention canbe used in immunoadjuvant therapy for the treatment of tumors (cancer).It is now well established that T cells recognize human tumor specificantigens. One group of tumor antigens, encoded by the MAGE, BAGE andGAGE families of genes, are silent in all adult normal tissues, but areexpressed in significant amounts in tumors, such as melanomas, lungtumors, head and neck tumors, and bladder carcinomas. DeSmet, C. et al.,Proc. Natl. Acad. Sci. USA, 93:7149 (1996). It has been shown thatcostimulation of T cells induces tumor regression and an antitumorresponse both in vitro and in vivo. Melero, I. et al., Nature Medicine.3:682 (1997); Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA, 94: 8099(1997); Lynch, D. H. et al., Nature Medicine, 3:625 (1997); Finn, O. J.and Lotze, M. T., J. Immunol., 21:114 (1998). The stimulatory compoundsof the invention can be administered as adjuvants, alone or togetherwith a growth regulating agent, cytotoxic agent or chemotherapeuticagent, to stimulate T cell proliferation/activation and an antitumorresponse to tumor antigens. The growth regulating, cytotoxic, orchemotherapeutic agent may be administered in conventional amounts usingknown administration regimes. Immunostimulating activity by thecompounds of the invention allows reduced amounts of the growthregulating, cytotoxic, or chemotherapeutic agents thereby potentiallylowering the toxicity to the patient.

K. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind to or complex with the polypeptides encoded by the genesidentified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art. All assays are commonin that they call for contacting the drug candidate with a polypeptideencoded by a nucleic acid identified herein under conditions and for atime sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g., on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labelledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London), 340, 245-246 (1989); Chien et al., Proc.Natl. Acad. Sci. USA, 8X, 9578-9582 (1991)] as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89, 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

In order to find compounds that interfere with the interaction of a geneidentified herein and other intra- or extracellular components can betested, a reaction mixture is usually prepared containing the product ofthe gene and the intra- or extracellular component under conditions andfor a time allowing for the interaction and binding of the two products.To test the ability of a test compound to inhibit binding, the reactionis run in the absence and in the presence of the test compound. Inaddition, a placebo may be added to a third reaction mixture, to serveas positive control. The binding (complex formation) between the testcompound and the intra- or extracellular component present in themixture is monitored as described above. The formation of a complex inthe control reaction(s) but not in the reaction mixture containing thetest compound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

L. Compositions and Methods for the Treatment of Immune Related Diseases

The compositions useful in the treatment of immune related diseasesinclude, without limitation, proteins, antibodies, small organicmolecules, peptides, phosphopeptides, antisense and ribozyme molecules,triple helix molecules, etc. that inhibit or stimulate immune function,for example, T cell proliferation/activation, lymphokine release, orimmune cell infiltration.

For example, antisense RNA and RNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4, 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

M. Anti-IL-17A/F Antibodies

In one embodiment, the present invention provides anti-IL-17A/Fantibodies which may find use herein as therapeutic and/or diagnosticagents. Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g., by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol, 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-IL-17A/F antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of a humanized anti-IL-17A/F antibody are contemplated.For example, the humanized antibody may be an antibody fragment, such asa Fab, which is optionally conjugated with one or more cytotoxicagent(s) in order to generate an immunoconjugate. Alternatively, thehumanized antibody may be an intact antibody, such as an intact IgG1antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of an IL-17A/F protein as describedherein. Other such antibodies may combine an IL-17A/F binding site witha binding site for another protein. Alternatively, an anti-IL-17A/IF armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptorsfor IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16),so as to focus and localize cellular defense mechanisms to theIL-17A/F-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express IL-17A/F. Theseantibodies possess an IL-17A/F-binding arm and an arm which binds thecytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricinA chain, methotrexate or radioactive isotope hapten). Bispecificantibodies can be prepared as full length antibodies or antibodyfragments (e.g., F(ab′)₂ bispecific antibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463.U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a V_(H)connected to a V_(L) by a linker which is tooshort to allow pairing between the two domains on the same chain.Accordingly, the V₁₁ and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H)domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fe region or a hinge region. In this scenario, the antibody willcomprise an Fe region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Feregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

8. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fe region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

9. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, agrowth inhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconj ugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomnonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin, and thetricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y, and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agentare made using a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC 1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

In one preferred embodiment, an anti-IL-17A/F antibody (full length orfragments) of the invention is conjugated to one or more maytansinoidmolecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-IL-17A/F Polypeptide Antibody-Maytansinoid Conjugates(Immunoconjugates)

Anti-IL-17A/F antibody-maytansinoid conjugates are prepared bychemically linking an anti-IL-17A/F antibody to a maytansinoid moleculewithout significantly diminishing the biological activity of either theantibody or the maytansinoid molecule. An average of 3-4 maytansinoidmolecules conjugated per antibody molecule has shown efficacy inenhancing cytotoxicity of target cells without negatively affecting thefunction or solubility of the antibody, although even one molecule oftoxinlantibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disufide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-IL-17A/F antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman etal., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-IL-17A/Fantibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPT, PAPTI,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-IL-17A/F antibodies. Examplesinclude At¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁸, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohcxane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-IL-17A/F antibodyand cytotoxic agent may be made, e.g., by recombinant techniques orpeptide synthesis. The length of DNA may comprise respective regionsencoding the two portions of the conjugate either adjacent one anotheror separated by a region encoding a linker peptide which does notdestroy the desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

10. Immunoliposomes

The anti-IL-17A/F antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

N. IL-17A/F Binding Oligopeptides

IL-17A/F binding oligopeptides of the present invention areoligopeptides that bind, preferably specifically, to an IL-17A/Fpolypeptide as described herein. IL-17A/F binding oligopeptides may bechemically synthesized using known oligopeptide synthesis methodology ormay be prepared and purified using recombinant technology. IL-17A/Fbinding oligopeptides are usually at least about 5 amino acids inlength, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids inlength or more, wherein such oligopeptides that are capable of binding,preferably specifically, to an IL-17A/F polypeptide as described herein.IL-17A/F binding oligopeptides may be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening oligopeptide libraries for oligopeptidesthat are capable of specifically binding to a polypeptide target arewell known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCTPublication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl.Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad.Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al.(1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. etal. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol., 2:668).

Tn this regard, bacteriophage (phage) display is one well knowntechnique which allows one to screen large oligopeptide libraries toidentify member(s) of those libraries which are capable of specificallybinding to a polypeptide target. Phage display is a technique by whichvariant polypeptides are displayed as fusion proteins to the coatprotein on the surface of bacteriophage particles (Scott, J. K. andSmith, G. P. (1990) Science 249: 386). The utility of phage display liesin the fact that large libraries of selectively randomized proteinvariants (or randomly cloned cDNAs) can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl.Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have beenused for screening millions of polypeptides or oligopeptides for oneswith specific binding properties (Smith, G. P. (1991) Current Opin.Biotechnol., 2:668). Sorting phage libraries of random mutants requiresa strategy for constructing and propagating a large number of variants,a procedure for affinity purification using the target receptor, and ameans of evaluating the results of binding enrichments. U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,689, and 5,663,143.

Although most phage display methods have used filamentous phage,lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024),T4 phage display systems (Ren, Z-J. et al. (1998) Gene 215:439; Zhu, Z.(1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z-J. etal. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efimov,V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems(Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217,22811257; U.S. Pat. No. 5,766,905) are also known.

Many other improvements and variations of the basic phage displayconcept have now been developed. These improvements enhance the abilityof display systems to screen peptide libraries for binding to selectedtarget molecules and to display functional proteins with the potentialof screening these proteins for desired properties. Combinatorialreaction devices for phage display reactions have been developed (WO98/14277) and phage display libraries have been used to analyze andcontrol bimolecular interactions (WO 98/20169; WO 98/20159) andproperties of constrained helical peptides (WO 98/20036). WO 97/35196describes a method of isolating an affinity ligand in which a phagedisplay library is contacted with one solution in which the ligand willbind to a target molecule and a second solution in which the affinityligand will not bind to the target molecule, to selectively isolatebinding ligands. WO 97/46251 describes a method of biopanning a randomphage display library with an affinity purified antibody and thenisolating binding phage, followed by a micropanning process usingmicroplate wells to isolate high affinity binding phage. The use ofStaphylococcus aureus protein A as an affinity tag has also beenreported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describesthe use of substrate subtraction libraries to distinguish enzymespecificities using a combinatorial library which may be a phage displaylibrary. A method for selecting enzymes suitable for use in detergentsusing phage display is described in WO 97/09446. Additional methods ofselecting specific binding proteins are described in U.S. Pat. Nos.5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these librariesare also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717,5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and5,723,323.

O. IL-17A/F Binding Organic Molecules

IL-17A/F binding organic molecules are organic molecules other thanoligopeptides or antibodies as defined herein that bind, preferablyspecifically, to an IL-17A/F polypeptide as described herein. IL-17A/Fbinding organic molecules may be identified and chemically synthesizedusing known methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). IL-17A/F binding organic molecules are usually less thanabout 2000 daltons in size, alternatively less than about 1500, 750,500, 250 or 200 daltons in size, wherein such organic molecules that arecapable of binding, preferably specifically, to an IL-17A/F polypeptideas described herein may be identified without undue experimentationusing well known techniques. In this regard, it is noted that techniquesfor screening organic molecule libraries for molecules that are capableof binding to a polypeptide target are well known in the art (see, e.g.,PCT Publication Nos. WO00/00823 and WO00/39585). IL-17A/F bindingorganic molecules may be, for example, aldehydes, ketones, oximes,hydrazones, semicarbazones, carbazides, primary amines, secondaryamines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols,ethers, thiols, thioethers, disulfides, carboxylic acids, esters,amides, ureas, carbamates, carbonates, ketals, thioketals, acetals,thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkylsulfonates, aromatic compounds, heterocyclic compounds, anilines,alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,thiazolidines, thiazolines, enamines, sulfonamides, epoxides,aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acidchlorides, or the like.

P. Screening for Anti-IL-17A/F Antibodies. IL-17A/F BindingOligopeptides and IL-17A/F Binding Organic Molecules with the DesiredProperties

Techniques for generating antibodies, oligopeptides and organicmolecules that bind to IL-17A/F polypeptides have been described above.One may further select antibodies, oligopeptides or other organicmolecules with certain biological characteristics, as desired.

The growth inhibitory effects of an anti-IL-17A/F antibody, oligopeptideor other organic molecule of the invention may be assessed by methodsknown in the art, e.g., using cells which express an IL-17A/Fpolypeptide either endogenously or following transfection with theIL-17A/F gene. For example, appropriate tumor cell lines andIL-17A/F-transfected cells may treated with an anti-IL-17A/F monoclonalantibody, oligopeptide or other organic molecule of the invention atvarious concentrations for a few days (e.g., 2-7) days and stained withcrystal violet or MTT or analyzed by some other colorimetric assay.Another method of measuring proliferation would be by comparing³H-thymidine uptake by the cells treated in the presence or absence ananti-IL-17A/F antibody, IL-17A/F binding oligopeptide or IL-17A/Fbinding organic molecule of the invention. After treatment, the cellsare harvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriate positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways known in the art.Preferably, the tumor cell is one that overexpresses an IL-17A/Fpolypeptide. Preferably, the anti-IL-17A/F antibody, IL-17A/F bindingoligopeptide or IL-17AF binding organic molecule will inhibit cellproliferation of an IL-17A/F-expressing tumor cell in vitro or in vivoby about 25-100% compared to the untreated tumor cell, more preferably,by about 30-100%, and even more preferably by about 50-100% or 70-100%,in one embodiment, at an antibody concentration of about 0.5 to 30μg/ml. Growth inhibition can be measured at an antibody concentration ofabout 0.5 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, wherethe growth inhibition is determined 1-10 days after exposure of thetumor cells to the antibody. The antibody is growth inhibitory in vivoif administration of the anti-IL-17A/F antibody at about 1 μg/kg toabout 100 mg/kg body weight results in reduction in tumor size orreduction of tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

To select for an anti-IL-17A/F antibody, IL-17A/F binding oligopeptideor IL-17A/F binding organic molecule which induces cell death, loss ofmembrane integrity as indicated by, e.g., propidium iodide (PI), trypanblue or 7AAD uptake may be assessed relative to control. A PI uptakeassay can be performed in the absence of complement and immune effectorcells. IL-17A/F polypeptide-expressing tumor cells are incubated withmedium alone or medium containing the appropriate anti-IL-17A/F antibody(e.g, at about 10 μg/ml), IL-17A/F binding oligopeptide or IL-17A/Fbinding organic molecule. The cells are incubated for a 3 day timeperiod. Following each treatment, cells are washed and aliquoted into 35mm strainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT®CellQuest software (Becton Dickinson). Those anti-IL-17A/F antibodies,IL-17A/F binding oligopeptides or IL-17A/F binding organic moleculesthat induce statistically significant levels of cell death as determinedby PI uptake may be selected as cell death-inducing anti-IL-17A/Fantibodies, IL-17A/F binding oligopeptides or IL-17A/F binding organicmolecules.

To screen for antibodies, oligopeptides or other organic molecules whichbind to an epitope on an IL-17A/F polypeptide bound by an antibody ofinterest, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. This assay can be usedto determine if a test antibody, oligopeptide or other organic moleculebinds the same site or epitope as a known anti-IL-17A/F antibody.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art. For example, the antibody sequence can bemutagenized such as by alanine scanning, to identify contact residues.The mutant antibody is initailly tested for binding with polyclonalantibody to ensure proper folding. In a different method, peptidescorresponding to different regions of an IL-17A/F polypeptide can beused in competition assays with the test antibodies or with a testantibody and an antibody with a characterized or known epitope.

Q. Pharmaceutical Compositions

The active IL-17A/F molecules of the invention (e.g., IL-17A/Fpolypeptides, anti-IL-17A/F antibodies, and/or variants of each) as wellas other molecules identified by the screening assays disclosed above,can be administered for the treatment of immune related diseases, in theform of pharmaceutical compositions.

Therapeutic formulations of the active IL-17A/F molecule, preferably apolypeptide or antibody of the invention, are prepared for storage bymixing the active molecule having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglubulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compounds identified by the screening assays disclosed herein can beformulated in an analogous manner, using standard techniques well knownin the art.

Lipofections or liposomes can also be used to deliver the IL-17A/Fmolecule into cells. Where antibody fragments are used, the smallestinhibitory fragment which specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variableregion sequences of an antibody, peptide molecules can be designed whichretain the ability to bind the target protein sequence. Such peptidescan be synthesized chemically and/or produced by recombinant DNAtechnology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,90:7889-7893 [1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active IL-17A/F molecules may also be entrapped in microcapsulesprepared, for example, by coaccrvation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations or the IL-17A/F molecules may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37□C, resulting in a loss of biological activity and possible changesin immunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

R. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat various immunerelated diseases and conditions, such as T cell mediated diseases,including those characterized by infiltration of inflammatory cells intoa tissue, stimulation of T-cell proliferation, inhibition of T-cellproliferation, increased or decreased vascular permeability or theinhibition thereof.

Exemplary conditions or disorders to be treated with the polypeptides,antibodies and other compounds of the invention, include, but are notlimited to systemic lupus erythematosis, rheumatoid arthritis, juvenilechronic arthritis, osteoarthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. Antibodies eitherdirectly or indirectly mediate tissue injury. Though T lymphocytes havenot been shown to be directly involved in tissue damage, T lymphocytesare required for the development of auto-reactive antibodies. Thegenesis of the disease is thus T lymphocyte dependent. Multiple organsand systems are affected clinically including kidney, lung,musculoskeletal system, mucocutaneous, eye, central nervous system,cardiovascular system, gastrointestinal tract, bone marrow and blood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that mainly involves the synovial membrane of multiple jointswith resultant injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against self TgG, with the resultantformation of immune complexes that attain high levels in joint fluid andblood. These complexes in the joint may induce the marked infiltrate oflymphocytes and monocytes into the synovium and subsequent markedsynovial changes; the joint space/fluid if infiltrated by similar cellswith the addition of numerous neutrophils. Tissues affected areprimarily the joints, often in symmetrical pattern. However,extra-articular disease also occurs in two major forms. One form is thedevelopment of extra-articular lesions with ongoing progressive jointdisease and typical lesions of pulmonary fibrosis, vasculitis, andcutaneous ulcers. The second form of extra-articular disease is the socalled Felty's syndrome which occurs late in the RA disease course,sometimes after joint disease has become quiescent, and involves thepresence of neutropenia, thrombocytopenia and splenomegaly. This can beaccompanied by vasculitis in multiple organs with formations ofinfarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, interstitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrheumatoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age. Its phenotype has somesimilarities to RA; some patients which are rheumatoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing spondylitis, Reiter'ssyndrome (reactive arthritis), arthritis associated with inflammatorybowel disease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8⁺ T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8⁺ T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class Imolecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8⁺T cells response.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin; likely this is induced by anactive inflammatory process. Scleroderma can be localized or systemic;vascular lesions are common and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICAM-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

Sjögren's syndrome is due to immune-mediated inflammation and subsequentfunctional destruction of the tear glands and salivary glands. Thedisease can be associated with or accompanied by inflammatory connectivetissue diseases. The disease is associated with autoantibody productionagainst Ro and La antigens, both of which are small RNA-proteincomplexes. Lesions result in keratoconjunctivitis sicca, xerostomia,with other manifestations or associations including biliary cirrhosis,peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including multiple sclerosis; idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome; and chronic inflammatory demyelinatingpolyneuropathy, are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. In MS there is evidence to suggest that diseaseinduction and progression is dependent on T lymphocytes. Multiplesclerosis is a demyelinating disease that is T lymphocyte-dependent andhas either a relapsing-remitting course or a chronic progressive course.The etiology is unknown; however, viral infections, geneticpredisposition, environment, and autoimmunity all contribute. Lesionscontain infiltrates of predominantly T lymphocyte mediated, microglialcells and infiltrating macrophages; CD4⁺ T lymphocytes are thepredominant cell type at lesions. The mechanism of oligodendrocyte celldeath and subsequent demyelination is not known but is likely Tlymphocyte driven.

Inflammatory and fibrotic lung disease, including eosinophilicpneumonia; idiopathic pulmonary fibrosis, and hypersensitivitypneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

Autoimmune or immune-mediated skin disease including bullous skindiseases, erythema multiforme, and contact dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

Transplantation associated diseases, including graft rejection andgraft-versus-host-disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Other diseases in which intervention of the immune and/or inflammatoryresponse have benefit are infectious disease including but not limitedto viral infection (including but not limited to AIDS, hepatitis A, B,C, D, E and herpes) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.

It has been demonstrated that some human cancer patients develop anantibody and/or T lymphocyte response to antigens on neoplastic cells.It has also been shown in animal models of neoplasia that enhancement ofthe immune response can result in rejection or regression of thatparticular neoplasm. Molecules that enhance the T lymphocyte response inthe MLR have utility in vivo in enhancing the immune response againstneoplasia. Molecules which enhance the T lymphocyte proliferativeresponse in the MLR (or small molecule agonists or antibodies thataffected the same receptor in an agonistic fashion) can be usedtherapeutically to treat cancer. Molecules that inhibit the lymphocyteresponse in the MLR also function in vivo during neoplasia to suppressthe immune response to a neoplasm; such molecules can either beexpressed by the neoplastic cells themselves or their expression can beinduced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

Additionally, inhibition of molecules with proinflammatory propertiesmay have therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatitis. The compounds of the present invention,e.g., polypeptides or antibodies, are administered to a mammal,preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerebral spinal,subcutaneous, intra-articular, intra synovial, intrathecal, oral,topical, or inhalation (intranasal, intrapulmonary) routes. Intravenousor inhaled administration of polypeptides and antibodies is preferred.

In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with a theimmunoadjuvant of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service, Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-oestrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

It may be desirable to also administer antibodies against other immunedisease associated or tumor associated antigens, such as antibodieswhich bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascularendothelial factor (VEGF). Alternatively, or in addition, two or moreantibodies binding the same or two or more different antigens disclosedherein may be coadministered to the patient. Sometimes, it may bebeneficial to also administer one or more cytokines to the patient. Inone embodiment, the IL-17A/F polypeptides are coadministered with agrowth inhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by an IL-17A/F polypeptide. However,simultaneous administration or administration first is alsocontemplated. Suitable dosages for the growth inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth inhibitory agent and the IL-17A/F polypeptide.

For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1mg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 mg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

S. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials (e.g., comprising an IL-17A/F molecule) useful forthe diagnosis or treatment of the disorders described above is provided.The article of manufacture comprises a container and an instruction.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for diagnosing or treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide or an antibody of the invention. An instruction or label on,or associated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

T. Diagnosis and Prognosis of Immune Related Disease

Cell surface proteins, such as proteins which are overexpressed incertain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, inflammatorybowel disorder, or another immune related disease, can be used asdiagnostics or prognostics.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by amplified or overexpressed genes (“marker gene products”).The antibody preferably is equipped with a detectable, e.g., fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the overexpressed gene encodes a cell surfaceprotein Such binding assays are performed essentially as describedabove.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Recombinant Expression of a Novel IL-17 Cytokine Identified asIL-17A/F

Human 293 Kidney Cells Transfection with cDNA Expression VectorsEncoding IL-17 and IL-17F

Human 293 kidney cells were transfected with equal amounts of plasmidsencoding the human IL-17, IL-17C and IL-17F genes, using a calciumphosphate precipitation procedure. For each 50%-80% confluent T-150flask, 50 μg of each plasmid was mixed to form a precipitate to layeronto cells. One day after transfection, 50:50 F12:DMEM containing 10%FCS, 5 mM L-glutamine, penicillin-streptomycin was removed and replacedwith serum-free PS24 media and cultured for an additional four days.After four days, conditioned media was collected centrifuged and sterilefiltered, prior to purification.

Purification of Recombinant IL-17A/F

A. Initial Fractionation Step 1:

Two and a half liters of recombinant IL-17A/F conditioned media fromhuman 293 kidney cell transient cultures was concentrated and dialyzedagainst 20 mM sodium acetate, pH 5.0, 1 mM sodium azide (Buffer A) usinga 10 kilodalton cutoff membrane to a volume of 480 milliliters, thenapplied to a Pharmacia HiLoad S Sepharose 26/10 column at 6 ml/min. Thecolumn was eluted with a linear gradient to 100% Buffer B (20 mM sodiumacetate, 1 M NaCl, 1 mM sodium azide, pH 5.0) at a rate of 1%/minutewith a flow rate of 6 ml/min collecting 12 ml fractions. SDS PAGEanalysis was performed on the fractions collected from this column.Proteins were revealed with silver staining. Molecular mass markers arelabeled for gel containing fractions 25-37 (FIG. 2). Fractions 31 and 32contained a protein with an apparent molecular mass of approximately 33kD consistent with IL-17A/F.

B. Purification of IL-17A/F:

Four ml of fraction 32 (FIG. 2) was acidified with 0.1% trifluoroaceticacid then applied at 0.5 ml/min to a Vydac C4 column equilibrated in0.1% trifluoroacetic acid (Buffer C) and gradient eluted to 100% BufferD (0.1% trifluoroacetic acid in 100% acetonitrile) with a three stepgradient (0-35% D over 10 minutes, 35-50% D over 35 minutes, 50-100% Dover 10 minutes). FIG. 2 shows the chromatograph of eluted proteinsmeasured at 214 nm and 280 nm. The acetonitrile step gradient isoverlain over the profile. Protein concentration of fraction 38 wasfound to be 0.536 mg/ml by amino acid analysis. Gels, blots, amino acidsequence and activity assays were run on this fraction.

Fraction 31 and the remaining volume of fraction 32, from the HiLoad SSepharose run were pooled and dialyzed against Buffer A for eight hoursusing a 10 kD cutoff membrane and passed through a 0.2 micron filter.This material was loaded on a Mono S column equilibrated in Buffer A ata flow rate of 1 ml/min and eluted with a three step gradient to 100%Buffer B (0-30% B over 10 column volumes, 30-75% B over 45 columnvolumes, 75-100% B over 10 column volumes) while collecting 1ml/fraction. Fractions 26-43 were assayed and protein concentrationswere determined by amino acid analysis. The concentration of fractions31, 32 and 33 were 0.258, 0.359 and 0.291 mg/ml respectively. Gels,blots, amino acid sequence, mass spectrophotometry and activity assayswere run primarily on fraction 32 and 33. Fractions generated bychromatography were assayed for IL-17 and IL-17F content through the useof Western blotting. One μg/ml of monoclonal antibody directed againsteither IL-17 or IL-17F was used to detect the presence of either IL-17or IL-17F in the samples.

Mass Spectrometry Analysis of IL-17A/F

The amino acid sequence and interchain disulfide bonds of matureIL-17A/F were determined by mass spectrometry analysis (see FIG. 4A;IL-17A/F heterodimeric polypeptide shown with interchain and intrachaindisulfide linkages). Two interchain disulfide linkages were detectedbetween IL-17F and IL-17 polypeptide chains [between residue 47_(IL-17F)and residue 129_(IL-17); and between residue 137_(IL-17F) and residue33_(IL-17), respectively (bold black lines in FIG. 4A). In addition, twointrachain disulfide links form in each of the homodimer polypeptidechains IL-17 [between residues 102 and 152; and between residues 107 to154] and IL-17F [between residues 94 and 144; and between residues 99and 146] (light black lines in FIG. 4A). The amino acids are numberedrelative to the initiating methionine in each precursor polypeptidechain (FIG. 4A). FIG. 4B shows a schematic of the IL-17A/F peptidefragments containing disulfide bonds between the IL-17 and the IL-17Fchain that would be anticipated by digestion of the IL-17A/F withtrypsin [IL-17A/F disulfide bond fragment #1 is designated as SEQ IDNO:7; TL-17AF disulfide bond fragment #2 is designated as SEQ ID NO:8,respectively]. The amino acids contained within these fragments areindicated and numbered relative to the initiating methionine of eachchain.

The calculated approximate molecular mass of these fragments that wouldbe observed by mass spectrometry is shown in FIG. 4B as 3410.58 Da and2420.05 Da [IL-17A/F disulfide bond fragment #1 and #2 respectively].Matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF) peptide mapping was performed (FIG. 4C). 55pmol of IL-17A/F in a buffer of 400 mM NaCl, 20 mM NaOAC buffer pH 5 wasdigested overnight at 37° C. with Promega sequencing grade trypsin.Matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF) was performed with delayed extraction inpositive ion reflectron mode using a 2′, 4′, 6′-trihydoxyacetophenonematrix. The resulting peptide map contained peaks with [M+H]+=2420.12 Dafor fragment #2 and 3410.60 Da for fragment #1, consistent with thedisulfide linked peptides (FIG. 4C). A second sample aliquot wasdigested at pH 8 following reduction of disulfide bonds withdithiothreitol and alkylation of sulfhydryl groups with iodoacetamide.The MALDI-TOF spectrum of this sample lacked the peaks in question,supporting their assignment as disulfide-linked. The non-reduced samplewas further characterized by liquid-chromatography electrosprayionization ion trap mass spectrometry (LC-ESI-MS) (FIG. 4D). The ionchromatograms represent (from top to bottom) the total ion chromatogram,reconstructed ion chromatogram (RIC) of IL-17A/F disulfide bond fragment#2 [M+2H]2+, and IL-17A/F disulfide bond fragment #1 [M+2H]3+. Peaksconsistent with both heterodimers were observed whereas no peaks abovebackground chemical noise were observed at the anticipated masses of thehomodimeric peptides thus indicating the absence of IL-17 or IL-17Fhomodimers. The composition of the disulfide-linked heterodimers wasthen confirmed by tandem mass spectrometry. Collision-induceddissociation of the doubly charged precursor at m/z 1210.9 correspondedto IL-17A/F disulfide bond fragment #2 and the triply charged precursorat m/z 1138.0 corresponds to IL-17A/F disulfide bond fragment #1.Predicted b- and y-ion series fragment peaks were observed in thecorresponding spectra.

Phage Library Screening for Antibodies that Bind to IL-17A/F

In order to identify antibodies which bind to IL-17A/F, a phage libraryof synthetic Fab antibodies was screened. Thirty four (34) independentclones encoding distinct Fab antibody sequences were identified. Whichwere able to mediate binding to IL-17A/F. The phage library of humanantibody sequences was prepared and screened for antigen specific Fab ina manner similar to that previously described (Gerstner, R. B. et al.,J. Mol. Biol., 321(5):851-62 (2002). Briefly, the humanized monoclonalantibody 4D5, an anti-HER2 antibody, was used as a scaffold to constructphage-displayed Fab libraries. These Fab are displayed on the phagemonovalently and/or divalently by fusion to a homodimerizable leucinezipper. To generate library diversity, we chose to randomize surfaceexposed heavy chain CDR residues that were also found to be highlydiverse in the Kabat database of natural antibody sequences and form acontiguous patch. Furthermore, we used site-directed mutagenesis withtailored degenerate codons to generate amino acid diversity thatmimicked the natural immune repertoire at each CDR site. First two CDRof heavy chain, H1 and H2, were allowed limited diversity of same lengthas Herceptin, whereas H3 is designed to have high degeneracy with lengthranged from 7 to 19. All antibodies generated from the initial libraryselection have the identical light chain. Full length IgG or Fab can begenerated by one-step cloning of the heavy chain variable domain intovectors providing the desired isotype specific constant region sequence.To further improve the affinity of binders from the heavy chain library,a second-step randomization of light chain CDRs can be employed. Theamino acid sequence of the region of the variable domain of the heavychains that contains the three (3) CDRs [H1-H3] from Fab that bindIL-17A/F are shown in FIG. 6. Shown is the alignment of a region of thepredicted amino acid sequence of 34 Fab clones that encode distinctantibody heavy chain sequences that are able to bind to IL-17A/F. Thethree heavy chain CDR regions are indicated as CDR-H1, CDR-H2 andCDR-H3, respectively are shaded. The corresponding SEQ ID NO for eachclone is as follows:

Clone #1=SEQ ID NO:9; Clone #2=SEQ ID NO:10; Clone #3=SEQ ID NO:11;Clone #4=SEQ ID NO: 12; Clone #5=SEQ ID NO:13; Clone #6=SEQ ID NO: 14;Clone #7=SEQ ID NO:15; Clone #8=SEQ ID NO:16; Clone #9=SEQ ID NO:17;Clone #10=SEQ ID NO:18; Clone #11=SEQ ID NO:19; Clone #12=SEQ ID NO:20;Clone #13=SEQ ID NO:21; Clone #14=SEQ ID NO:22; Clone #15=SEQ ID NO:23;Clone #16=SEQ ID NO:24; Clone #17=SEQ ID NO:25; Clone #18=SEQ ID NO:26;Clone #19=SEQ 1D NO:27; Clone #20=SEQ ID NO:28; Clone #21=SEQ ID NO:29;Clone #22=SEQ ID NO:30; Clone #23=SEQ ID NO:31; Clone #24=SEQ ID NO:32;Clone #25=SEQ ID NO:33; Clone #26=SEQ ID NO:34; Clone #27=SEQ ID NO:35;Clone #28=SEQ ID NO:36; Clone #29=SEQ ID NO:37; Clone #30=SEQ ID NO:38;Clone #31=SEQ ID NO:39; Clone #32=SEQ ID NO:40; Clone #33=SEQ ID NO:41;Clone #34=SEQ ID NO:42, respectively.

In addition, the corresponding encoding DNA sequences for each of thethirty four (34) clones is shown in Table 7 below (SEQ ID NO:43 to SEQID NO:76, respectively).

TABLE 7 SEQ ID NO: 43:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGATTCCGCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTGGGATTACTCCTTATAGCGGTTATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAAAGAGGCCCGCGAGGGCTACGACGTCGGCTACGCTATGGACTACTGGGGTCAASEQ ID NO: 44:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGATTCCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTGAAATTTCTCCTCCTGGCGGCGATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTCTCTTGTGGTGGTGGGACGGGGCTATGGACTACTGGGGTCAA SEQ ID NO: 45:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAATACTTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTGTTATTACTCCTTATGGCGGTGCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAGAGAGAGTATGTGGAGTAAGTTCGACTACTGGGGTCAA SEQ ID NO: 46:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATAGTTCTGCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTATATTACTCCTGATAACGGTGATACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAATAGCTGAGGATACTGCCGTCTATTATTGTGCTCGCGGCCACGGCAACTTCTACGGTACCTGGGCGGCTATGGACTACTGGGGTCAASEQ ID NO: 47:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGGTTCTGATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTATATTAATCCTTATGGCGGTTCTACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGCGTACGAGATGTGGTACGTTATGGACTACTGGGGTCAA SEQ ID NO: 48:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAATTCCTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTGTTATTACTCCTTCTAGCGGTTCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGGTCTTCCCCGACATCGGGGACTGCAGCAACGCCTACTGCTACGCTATGGACTACTGGGGTCAA SEQ ID NO: 49:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAGTACTTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTATAGCGGTTATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGGTGGGGTGGGGGGACTCGTACGCTATGGACTACTGGGGTCAASEQ ID NO: 50:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGGTTCTTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTGGGATTTATCCTTATGACGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGGCCGAGGGCCTGTACCAGTCCGGGATCTACGACGCGGGTATGGACTACTGGGGTCAA SEQ ID NO: 51:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAGTTACTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTATCCTGCTGACGGTGCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGGGTCCTACTTCGGGGGCTACGATATGGACTACTGGGGTCAA SEQ ID NO: 52:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATGATTCTGATATAACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTATTATTTATCCTTATGACGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAGAAGCAACCTGGACAACAACTTGTTCGACTACTGGGGTCAA SEQ ID NO: 53:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATGGTTACTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTGATATTAATCCTAATGGCGGTTCTACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGCCTACCGGTGCGGCGGGCTCGCCGACTGGGCCGGGGCTATGGACTACTGGGGTCAASEQ ID NO: 54:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGGTTCTTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTATTATTACTCCTTCTGGCGGTAATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGGTCTTCGCCGTGTCGACCGCCGGCTACCCCTGGGTTATGGACTACTGGGGTCAASEQ ID NO: 55:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATTCTTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTCTATTACTCCTTATAACGGTAATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCAGGGGGGAGTCCGACGAGGCCTACGCCGCGGTTATGGACTACTGGGGTCAASEQ ID NO: 56:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTAGTTCCGATATACACTGGGTGCGTCAGGCCCCGGGTAGGGCCTGGAATGGGTTGGTACTATTAATCCTGCTAGCGGTTCTACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGGCGCCAACAGCAGCTTCTACGCGCTCCAGTACGTTATGGACTACTGGGGTCAASEQ ID NO: 57:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATAATTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTGGATTTCTCCTTATAGCGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGACCCTCTTCTACGACAAGGACCAGTACTCCTACGTTATGGACTACTGGGGTCAASEQ ID NO: 58:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTAGTTCTTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTATAGCGGTTATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGGGGCTCCTGCGGTGGGGCTACGCTATGGACTACTGGGGTCAASEQ ID NO: 59:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATAATGGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTGGATTACTCCTACTAGCGGTTATACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGACGGGGACACCTGGAAGTGGGACGCCCCGTACGTTATGGACTACTGGGGTCAASEQ ID NO: 60:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAATACTTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTATAGCGGTTATACTGACTATGCCGATAGCGTCAAGGACCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGAGATCTTGCTGGACTACGGTTCCGCGGGCTACGCTATGGACTACTGGGGTCAASEQ ID NO: 61:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAGTACCTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTGTTATTACTCCTACTAACGGTTCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGAGGTGTGGTGGTGGGGCGACGGCCACGGCTACGTTATGGACTACTGGGGTCAASEQ ID NO: 62:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTAGTTCTGCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTGGGATTACTCCTGCTAGCGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCGCCCGGCGGGGTGTTCGTCGACGGCGGGGTTATGGACTACTGGGGTCAASEQ ID NO: 63:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATAGTACTGATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTAGGATTAATCCTTCTGGCGGTTCTACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTACCAGCGCGTACACCACGTGGGCGGTCGACTGGTTCATCGGCTACGTTATGGACTACTGGGGTCAA SEQ ID NO: 64:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGGTTACGGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTCTAACGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTCGCGTCAGCTACTACGTCTACAGGCACGACTGGGTCAGGGGCTACGTTATGGACTACTGGGGTCAA SEQ ID NO: 65:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATACCTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTGTTATTACTCCTTATGGCGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAGAGACGGGGGCTTCTTCGATTACTGGGGTCAA SEQ ID NO: 66:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGATTCCTCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTTTATTTATCCTACTAGCGGTTCTACTACTATGCCAATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCACGTGCCTCGTACGGGGTGAGCAAGTGGACCTTTGACTACTGGGGTCAASEQ ID NO: 67:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGGTTACGGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTCTAACGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCATACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTCGCGTCAGCTACTACGTCTACAGGACGACTGGGTCAGGGGCTACGTTATGGACTACTGGGGTCAA SEQ ID NO: 68:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGGTACTTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTATAGCGGTTATACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAGAGAGGCCCGCTCCTCGTTGAGCGCGGACTACGCTATGGACTACTGGGGTCAASEQ ID NO: 69:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATAATTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTTATAGCGGTTATACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTGAGTCCGGCTTCTCCGCGTGCAACACGCGGGCGTACGCTATGGACTACTGGGGTCAASEQ ID NO: 70:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGATTCTTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTCTATTACTCCTTATAACGGTAATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCAGGGGGGAGTCCGACGAGGCCTACCCCGCGGTTATGGACTACTGGGGTCAASEQ ID NO: 71:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTAGTACCGCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTACTCCTTATGACGGTTATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACTAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTACGTGGTTCACGCTGGCCTCGGCTATGGAACTACTGGGGTCAASEQ ID NO: 72:TTGTCCTGTGCAGCTTCTGGCTTCACCATTACTGGTAATGGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTTCTCCTACTAACGGTTCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTAGGGTCGACTACCAGGTCTACCACGACCGCTTCGAGGAGGGGTACGCTATGGACTACTGGGGTCAA SEQ ID NO: 73:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATAGTTATTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTGGATTTCTCCTGATAACGGTGCTACTAACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTAAGTTCTGGGGCTGGGACTGGGGGGGTATGGACTACTGGGGTCAASEQ ID NO: 74:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGATTCTTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTGATATTACTCCTACTGACGGTTATACTGACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTAACTTGATGTGGTGGGACTCGTCGGCTATGGACTACTGGGGTCAASEQ ID NO: 75:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAGTGATTCTGGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGGTTTTATTTATCCTAATGGCGGTTCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGTATGTCGTTGATCGGGTTCTCGTACGCTATGGACTACTGGGGTCAASEQ ID NO: 76:TTGTCCTGTGCAGCTTCTGGCTTCACCATTAATAGTACCTGGATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCTTGGATTAATCCTTATAACGGTTCTACTTACTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCAAGAGACTTGTACGACTACGACATCGGCTTCGACTACTGGGGTCAA

Cell-Based Assays—IL-17A/F Induces the Production of IL-8 and IL-6

Fractions isolated from the Vydac C4 purification step described above(FIG. 3) were assayed for the ability of IL-17A/F to induce theproduction of IL-8. Fractions were tested by incubation with TK-10 cellsfor 24 hours (0.033 microliters fraction/ml of cell culture media).Conditioned media was then collected and IL-8 and IL-6 concentrationmeasurements were performed on each fraction by ELISA. Fraction 38 wasfound to have robust activity. Protein concentration of fraction 38 wasfound to be 0.536 mg/ml by amino acid analysis. Gels, blots, amino acidsequence and activity assays were run on this fraction (FIG. 3).Alternatively, fraction 31 and the remaining volume of fraction 32, fromthe HiLoad S Sepharose run were pooled and dialyzed against Buffer A foreight hours using a 10 kD cutoff membrane and passed through a 0.2micron filter. This material was loaded on a Mono S column equilibratedin Buffer A at a flow rate of 1 ml/min and eluted with a three stepgradient to 100% Buffer B (0-30% B over 10 column volumes, 30-75% B over45 column volumes, 75-100% B over 10 column volumes) while collecting 1ml/fraction. Fractions 26-43 were assayed and protein concentrationswere determined by amino acid analysis. Pure IL-17A/F was identified infractions 31-33 as a single protein with apparent molecular mass of30-35 kD. The concentrations of fractions 31, 32 and 33 were 0.258,0.359 and 0.291 mg/ml respectively. Gels and protein sequence analysisshowed this material to be identical to IL-17A/F purified by C4 column(above). Dose response curves comparing IL-8 and IL-6 induction byIL-17A/F, IL-17 and IL-17F are shown in FIG. 5. IL-17A/F, IL-17 andIL-17F were incubated with TK-10 cells at the indicated concentrationsfor 24 hours. TK-10 conditioned media was collected and analyzed by IL-8ELISA and 11-6 ELISA.

DISCUSSION

Co-expression of mRNA for IL-17 and IL-17F leads to the secretion of anovel protein species that is able to bind with both certain antibodiesthat are capable of binding to IL-17 and certain antibodies that arecapable of binding to IL-17F. This novel protein species is designatedherein as interleukin-17A/F (IL-17A/F). This species is not observedwhen human kidney 293 cells are made to express either IL-17 or IL-17Fin isolation. Conditioned media from transfected cells wasimmunoprecipitated (IP) utilizing antibodies that are able to recognizeIL-17 (lanes 1-5) or IL-17F (lanes 6-10) as shown in FIG. 1A and FIG.1B. Immunoprecipitated proteins were then resolved by Western blotanalysis and blotted with antibodies to IL-17 (FIG. 1A) or IL-17F (FIG.1B). Detection of IL-17A/F is indicated in lane 8 of FIG. 1A and in lane3 of FIG. 1B by the presence of 11-17 in dimeric complex with IL-17F.The molecular mass of this species, as determined by non-reducingSDS-PAGE is approximately 30-35 kD, consistent with the species beingcomprised of one molecule of IL-17 and one molecule of IL-17F joined bycovalent linkage. The existence of this new species (IL-17A/F) can alsobe recognized as protein of electrophoretic mobility that is distinctfrom that observed when either IL-17 or IL-17F is expressed inisolation. As such, this new species can also be visualized without theuse of antibodies through the use of other protein detection methodssuch as conventional protein staining techniques.

The existence of a novel protein species produced by co-expression ofIL-17 and IL-17F was also observed by resolving the secreted proteinspresent in conditioned media with reverse phase chromatography.Comparison of the protein fractions observed from the secreted proteinsproduced by cells co-expressing IL-17 and IL-17F with the patternsobserved with cells producing either IL-17 or IL-17F revealed thepresence of an additional protein species. This protein species,IL-17A/F, was purified and isolated to homogeneity by columnchromatography (FIGS. 2 and 3).

Purified protein ran as a single band of approximately 30-35 kD asdetermined by non-reducing SDS-PAGE (FIG. 3A). However, under reducingconditions two clearly distinct bands were revealed with an apparentmolecular mass of approximately 15-18 kD (not shown). Thus, IL-17A/F isa covalent dimer. An independent means of assessing the composition ofthe novel protein, N-terminal peptide sequence analysis, also clearlyindicated that the isolated IL-17A/F contains both IL-17 and IL-17Fpeptides (FIG. 3B). The detected peptide sequences are identical tosequence contained within the N-terminal end of IL-17 and IL-17F (FIG.3C). Western Blot analysis indicated that this novel protein species isalso able to interact both with an antibody that is able to bind toIL-17 and with an antibody that is able to bind to IL-17F. Each of theseobservations and the distinct molecular mass of the novel isolatedprotein species suggest that the isolated protein IL-17A/F is a novelprotein species comprised of a covalent association of IL-17 and IL-17F.

The existence and location of the disulfide bonds that link the IL-17and IL-17F chains of IL-17A/F were further characterized by use of massspectrometry. The position of disulfide linkages within IL-17A/F isshown in schematic FIG. 4A. Two interchain disulfide bonds link theIL-17 and IL-17F chains in IL-17A/F. Digestion of IL-17A/F with trypsinwould be expected to produce two distinct peptide fragments containingthe interchain disulfide bonds (IL-17A/F disulfide bond fragment #1 and#2; SEQ ID NOs:7 and 8, respectively. These peptides are shownschematically (FIG. 4B) together with the respective predicted molecularmass. These peptides were observed by Marix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI-TOF) (FIG.4C) and by liquid-chromatography electrospray ionization ion trap massspectrometry (LC-ESI-MS) (FIG. 4D). Peptide peaks corresponding tohomodimers of IL-17 or IL-17F were not detected, indicating that thepurified IL-17A/F was comprised of covalent heterodimers of IL-17 andIL-17F chains and did not contain detectable levels of homodimers ofeither IL-17 or IL-17F.

In addition, antibodies which bind to IL-17A/F have been identified byscreening a phage library of synthetic Fab antibodies. Thirty four (34)independent clones encoding distinct Fab antibody sequences wereidentified. Which were able to mediate binding to IL-17A/F. The aminoacid sequence of the region of the variable domain of the heavy chainsthat contains the three (3) CDRs [H1-H3] from Fab that bind IL-17A/F areshown in FIG. 6. Shown is the alignment of a region of the predictedamino acid sequence of 34 Fab clones that encode distinct antibody heavychain sequences that are able to bind to IL-17A/F. The three heavy chainCDR regions are indicated as CDR-H1, CDR-H2 and CDR-H3, respectively arehighlighted in yellow. The corresponding amino acid sequences for eachof the thirty four (34) clones are identified as SEQ ID NOs:9-42. Inaddition, the corresponding encoding DNA sequences for each of theidentified thirty four (34) clones is shown in Table 7 below (SEQ IDNO:43 to SEQ ID NO:76, respectively). Thus, specific antibodies whichbind selectively to the novel heterodimeric complex of IL-17A/F havebeen identified which may serve to modulate the activity of this novelcytokine.

IL-17A/F was analyzed for ability to stimulate a proinflammatoryresponse using the TK-10 human kidney cell line (FIG. 5). This cell lineresponds to both IL-17 and IL-17F by production of IL-8. IL-17A/F alsorobustly induced IL-8 production in this cell line (FIG. 5A).Interestingly, IL-17A/F was observed to have a unique potency thatdiffers from that of either IL-17 or IL-17F. The difference in activitydiffers from IL-17 and IL-17F by roughly an order of magnitude in eachcase. The substantially greater activity of IL-17A/F than IL-17F in thisassay suggests that IL-17A/F may comprise a critical component of thecytokine activity resulting from the IL-17F gene product. This uniquepotency may enable the molecule to possess distinct range of actions invivo. IL-17A/F also induced production of IL-6 from this cell line (FIG.5B). Additionally, it is likely that IL-17A/F may possess additionalcharacteristics not present in either IL-17 or IL-17F as a result of itsnovel heterodimeric composition that may alter the kinetics andutilization of receptor subunits in vivo, resulting in unique biologicalconsequences.

Example 2 Identification of a Novel IL-17 Cytokine Produced in ActivatedHuman T Cells

A novel human IL-17 cytokine (herein identified as human IL-17A/F) isherein described for the first time as being naturally produced inactivated human T-lymphocyte cells. Isolation and activation of humanT-lymphocyte cells was performed and IL-17A/F production was detectedand quantitatively measured by IL-17A/F ELISA as demonstrated below:

Isolation and Activation of Human T-Cells

Heparinized (0.5 ml/50 cc) freshly-drawn human blood from a normalhealthy donor was diluted 1:1 with physiological saline, then layeredonto LSM Lymphcyte Separation Media (ICN) and centrifuged as recommendedby the manufacturer (ICN). Recovered mononuclear lymphocytes were platedin tissue culture flasks in complete RPMI (RPMI, 10% FCS, 2 mML-Glutamine, Penicillin/Streptomycin (GIBCO)), for one hour at 37degrees C. to deplete mnonocytes. Culture supernates were centrifuged topellet the remaining cells. Human T lymphocytes were then isolated bynegative selection using a CD4+ T cell isolation kit (MACS). To activatethe isolated T lymphocytes, tissue culture flasks were coated with 5ug/ml each of anti-CD3 (BD Bioscience) and anti-CD28 (BD Bioscience) inPBS overnight at 4 degrees C. After removing the coat media, isolatedhuman T lymphocytes were plated in complete RPMI at an approximatedensity of 2 million cells per milliliter of media. Samples of mediawere collected at various time points following plating and assayed forIL-17A/F by ELISA. Non-activated control supernates were collected fromcell supernatants from flasks not coated with anti-CD3 and anti-CD28.

ELISA Measurement of Human IL-17A/F Production in Anti-CD3/Anti-CD28Activated Human T-Cells

Human IL-17A/F levels were measured by ELISA. Mouse anti-human IL-17 wasdiluted in coat buffer (0.05 M sodium carbonate buffer, pH 9.6) andcoated on 96-well microtiter plates (Nunc), for 12-15 hours at 2-8IC.All subsequent steps were performed at room temperature. Non-specificbinding was blocked by emptying the wells and adding block buffer (PBS,0.5% BSA, 10 ppm Proclin 300). After a 1 hour incubation, the wells werewashed with wash buffer (PBS, 0.05% Tween 20, 10 ppm Proclin 300). HumanIL-17A/F reference standards and samples, diluted in assay buffer (PBS,0.5% BSA, 0.05% Tween 20, 10 ppm Proclin 300) were then added. Followinga 2-hour incubation, the wells were washed with wash buffer.Biotinylated mouse anti-human IL-17F, diluted in assay buffer, was addedand allowed to incubate for L hour. After washing the plates with washbuffer, Streptavidin-HRP (horseradish peroxidase) (Amersham), diluted inassay buffer, was added and allowed to incubate for 1 hour. Afterwashing the plates with wash buffer, the substrate solution, TMB (tetramethyl benzidine)-Peroxidase (R & D Systems) was added. Colordevelopment was stopped by adding 2 N sulphuric acid. The plates werethen read on a microtiter plate reader (SLT) at 450 nm with a subtractedblank at 540 nm. A four-parameter curve-fitting program was used togenerate a standard curve, and sample concentrations were derived byinterpolation from the linear portion of the curve. IL-17A and IL-17Fwere included as controls in the ELISA to illustrate the assayspecificity for IL-17A/F (FIG. 12).

Results:

The results of ELISA measurements of IL-17A/F production is shown inFIG. 11. These studies demonstrate the production of a novel cytokineIL-17A/F from anti-CD3/anti-CD28 activated human T lymphocyte cellscompared to non-activated human T-cells wherein no production ofIL-17A/F was detected. These results show for the first time the naturaloccurrence of a novel cytokine which is produced and released inresponse to the activation of human T lymphocytes. In addition, thespecificity of the ELISA assay was demonstrated by observing nearlyequivalent quantities of IL-17A/F in three samples (#31-#33) whenassayed in parallel. Negligible amounts of IL-17A or IL-17F weredetected in this IL-17A/F specific ELISA (FIG. 12).

The studies described herein in both Example 1 and 2 establish thatrecombinant human IL-17A/F is a distinctly new cytokine, distinguishablefrom human IL-17 and IL-17F in both protein structure and in cell-basedactivity assays. Through the use of purified recombinant human IL-17A/Fas a standard, a human IL-17AF-specific ELISA has been developed (shownin FIG. 11). Through the use of this specific ELISA, the inducedexpression of human IL-17A/F was detected, confirming that IL-17A/F isnaturally produced from activated human T cells in culture. Hence,IL-17A/F is a distinctly new cytokine, detectable as a natural productof isolated activated human T cells, whose recombinant form has beencharacterized, in both protein structure and cell-based assays, as to bedifferent and distinguishable from related cytokines.

This new cytokine can act to modulate the activity of IL-17 in vivo,acting as a competitive inhibitor to binding sites for IL-17 or otherrelated cytokines. IL-17A/F can also modulate the activity of otherrelated cytokines by down regulation of binding sites for itself and/orbinding sites for other related cytokines. IL-17A/F can exhibit activitythrough intracellular adapters or signaling molecules which act toaffect its own signaling activity or that of other related cytokines.IL-17A/F has the ability to affect the pairing of receptors andco-receptors found at the surface of cells or within the intracellularcompartment.

Thus, these studies provide and identify a novel immune stimulant (i.e.IL-17A/F) that can boost the immune system to respond to a particularantigen that may not have been immunologically active previously. Assuch, the newly identified immune stimulant has important clinicalapplications. Other known immune stimulants such as IL-12 have beenidentified. [see Gubler et al. PNAS 88, 4143 (1991)]. In a recent cancervaccine trial, researchers from the University of Chicago and GeneticsInstitute (Cambridge, Mass.) have relyed upon the immune stimulatoryactivity of IL-12, for the treatment of melanoma. [Peterson et al.Journal of Clinical Oncology 21 (12). 2342-48 (2003)] They extractedcirculating white blood cells carrying one or more markers of melanomacells, isolated the antigen, and returned them to the patients. Normallypatients would not have an immune response to his or her own humanantigens. The patients were then treated with different doses of IL-12,an immune stimulant capable of inducing the proliferation of T cellsthat have been co-stimulated by dendritic cells. Due to the immunestimulatory effect of IL-12, the treatment provided superior results incomparison to earlier work, where patients' own dendritic cells wereprepared from peripheral blood mononuclear cells (PBMCs), treated withantigens, then cultured in vitro and returned to the patient tostimulate anti-cancer response. [Thurner et al. J. Exp. Med. 190 (11),1669-78 (1999)] Likewise, this novel IL-17A/F cytokine or agoniststhereof, would therefore find practical utility as an immune stimulant.Whereas molecules which inhibit IL-17A/F activity (antagonists) would beexpected to find practical utility when an inhibition of the immuneresponse is desired, such as in autoimmune diseases.

Thus, antibodies to this new cytokine which either mimic (agonistantibodies) or inhibit (antagonist antibodies) the immunologicalactivities of IL-17A/F would possess therapeutic qualities. Smallmolecules which act to inhibit the activity of this novel cytokine wouldalso have potential therapeutic uses.

Example 3 Use of IL-17A/F as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingIL-17A/F as a hybridization probe.

DNA comprising the coding sequence of full-length or mature IL-17A/F asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of IL-17A/F) inhuman tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled IL-17A/F-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence 11_-17A/F can then be identified usingstandard techniques known in the art.

Example 4 Expression of IL-17A/F in E. coli

This example illustrates preparation of an unglycosylated form ofIL-17A/F polypeptides by recombinant expression in E. coli.

The DNA sequence encoding an IL-17A/F polypeptide is initially amplifiedusing selected PCR primers. The primers should contain restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector. A variety of expression vectors may beemployed. An example of a suitable vector is pBR322 (derived from E.coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyHis leader (including the first six STII codons, polyHissequence, and enterokinase cleavage site), the IL-17A/F polypeptidecoding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized IL-17A/F protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

IL-17A/F polypeptides may be expressed in E. coli in a poly-His taggedform, using the following procedure. The DNA encoding an IL-17A/Fpolypeptide is initially amplified using selected PCR primers. Theprimers will contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector, and otheruseful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences are then ligated into an expression vector, which isused to transform an E. coli host based on strain 52 (W3110 fuhA(tonA)Ion galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LBcontaining 50 mg/ml carbenicillin at 30° C. with shaking until anO.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold intoCRAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodiumcitrate-2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffieldhycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v)glucose and 7 mM MgSO₄) and grown for approximately 20-30 hours at 30°C. with shaking. Samples are removed to verify expression by SDS-PAGEanalysis, and the bulk culture is centrifuged to pellet the cells. Cellpellets are frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteinc residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded IL-17A/F polypeptide are pooledand the acetonitrile removed using a gentle stream of nitrogen directedat the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtrationusing G25 Superfine (Pharmacia) resins equilibrated in the formulationbuffer and sterile filtered.

Example 5 Expression of IL-17A/F in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof IL-17A/F polypeptides by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the IL-17A/F DNA is ligated intopRK5 with selected restriction enzymes to allow insertion of theIL-17A/F DNA using ligation methods such as described in Sambrook etal., supra. The resulting vector is called pRK5-IL-17A/F.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-IL-17A/F DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 ρCi/ml ³⁵S-cysteine and 200 ρC/ml ³⁵S-methionine. After a12 hour incubation, the conditioned medium is collected, concentrated ona spin filter, and loaded onto a 15% SDS gel. The processed gel may bedried and exposed to film for a selected period of time to reveal thepresence of the IL-17A/F polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

In an alternative technique, IL-17A/F may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-IL-17A/F DNA isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing the expressed IL-17A/F polypeptide canthen be concentrated and purified by any selected method, such asdialysis and/or column chromatography.

In another embodiment, IL-17A/F polypeptides can be expressed in CHOcells. The pRK5-IL-17A/F can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of the IL-17A/F polypeptide, the culture mediummay be replaced with serum free medium. Preferably, the cultures areincubated for about 6 days, and then the conditioned medium isharvested. The medium containing the expressed IL-17A/F polypeptide canthen be concentrated and purified by any selected method.

Epitope-tagged IL-17A/F may also be expressed in host CHO cells. TheIL-17A/F may be subcloned out of the pRK5 vector. The subclone insertcan undergo PCR to fuse in frame with a selected epitope tag such as apoly-His tag into a Baculovirus expression vector. The poly-His taggedIL-17A/F insert can then be subcloned into a SV40 driven vectorcontaining a selection marker such as DHFR for selection of stableclones. Finally, the CHO cells can be transfected (as described above)with the SV40 driven vector. Labeling may be performed, as describedabove, to verify expression. The culture medium containing the expressedpoly-His tagged IL-17A/F can then be concentrated and purified by anyselected method, such as by Ni²⁺-chelate affinity chromatography.

IL-17A/F polypeptides may also be expressed in CHO and/or COS cells by atransient expression procedure or in CHO cells by another stableexpression procedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g., extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, C112 and CH2domains, and/or as a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used in expression in CHOcells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min, at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 6 Expression of IL-17A/F in Yeast

The following method describes recombinant expression of IL-17A/Fpolypeptides in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of IL-17A/F from the ADH2/GAPDH promoter. DNAencoding the IL-17A/F polypeptide and the promoter is inserted intosuitable restriction enzyme sites in the selected plasmid to directintracellular expression of the IL-17A/F polypeptide. For secretion, DNAencoding IL-17A/F can be cloned into the selected plasmid, together withDNA encoding the ADH2/GAPDH promoter, a native IL-17A/F signal peptideor other mammalian signal peptide, or, for example, a yeast alpha-factoror invertase secretory signal/leader sequence, and linker sequences (ifneeded) for expression of IL-17A/F.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant IL-17A/F polypeptides can subsequently be isolated andpurified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing the IL-17A/F polypeptidemay further be purified using selected column chromatography resins.

Example 7 Expression of IL-17A/F in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of IL-17A/Fpolypeptides in Baculovirus-infected insect cells.

The sequence coding for IL-17A/F is fused upstream of an epitope tagcontained within a Baculovirus expression vector. Such epitope tagsinclude poly-His tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVLI393 (Novagen). Briefly, thesequence encoding EL-17A/F or the desired portion of the coding sequenceof IL-17A/F such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-His tagged IL-17A/F can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted Hisio-tagged IL-17A/F are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) IL-17A/Fcan be performed using known chromatography techniques, including forinstance, Protein A or Protein G column chromatography.

Example 8 Preparation of Antibodies that Bind IL-17A/F

This example illustrates preparation of monoclonal antibodies which canspecifically bind IL-17A/F.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified IL-17A/F polypeptides, fusion proteinscontaining IL-17A/F polypeptides, and cells expressing recombinantIL-17A/F polypeptides on the cell surface. Selection of the immunogencan be made by the skilled artisan without undue experimentation.

Mice, such as BALB/c, are immunized with the IL-17A/F immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-IL-17A/F antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of IL-17A/F. Three to four days later, the mice are sacrificedand the spleen cells are harvested. The spleen cells are then fused(using 35% polyethylene glycol) to a selected murine myeloma cell linesuch as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstIL-17A/F. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against IL-17A/F is within the skill inthe art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic BALB/c mice to produce ascites containing the anti-IL-17A/Fmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 9 Purification of IL-17A/F Polypeptides Using SpecificAntibodies

Native or recombinant IL-17A/F polypeptides may be purified by a varietyof standard techniques in the art of protein purification. For example,pro-IL-17A/F polypeptide, mature IL-17A/F polypeptide, or pre-IL-17A/Fpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the IL-17A/F polypeptide of interest. Ingeneral, an immunoaffinity column is constructed by covalently couplingthe anti-IL-17A/F polypeptide antibody to an activated chromatographicresin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification ofIL-17A/F polypeptide by preparing a fraction from cells containingIL-17A/F polypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble IL-17A/Fpolypeptide containing a signal sequence may be secreted in usefulquantity into the medium in which the cells are grown.

A soluble IL-17A/F polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of IL-17A/F polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/IL-17A/F polypeptidebinding (e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), andIL-17A/F polypeptide is collected.

Example 10 Drug Screening

This invention is particularly useful for screening compounds by usingIL-17A/F polypeptides or binding fragment thereof in any of a variety ofdrug screening techniques. The IL-17A/F polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing theIL-17A/F polypeptide or fragment. Drugs are screened against suchtransformed cells in competitive binding assays. Such cells, either inviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between IL-17A/Fpolypeptide or a fragment and the agent being tested. Alternatively, onecan examine the diminution in complex formation between the IL-17A/Fpolypeptide and its target cell or target receptors caused by the agentbeing tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect an IL-17A/F polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an IL-17A/F polypeptide or fragment thereof and assaying (i) forthe presence of a complex between the agent and the IL-17A/F polypeptideor fragment, or (ii) for the presence of a complex between the IL-17A/Fpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the IL-17A/F polypeptide or fragmentis typically labeled. After suitable incubation, free IL-17A/Fpolypeptide or fragment is separated from that present in bound form,and the amount of free or uncomplexed label is a measure of the abilityof the particular agent to bind to IL-17A/F polypeptide or to interferewith the IL-17A/F polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to an IL-17A/F polypeptide, the peptide testcompounds are reacted with IL-17A/F polypeptide and washed. BoundIL-17A/F polypeptide is detected by methods well known in the art.Purified IL-17A/F polypeptide can also be coated directly onto platesfor use in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding IL-17A/Fpolypeptide specifically compete with a test compound for binding toIL-17A/F polypeptide or fragments thereof. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with IL-17A/F polypeptide.

Example 11 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., an IL-17A/Fpolypeptide) or of small molecules with which they interact, e.g.,agonists, antagonists, or inhibitors. Any of these examples can be usedto fashion drugs which are more active or stable forms of the IL-17A/Fpolypeptide or which enhance or interfere with the function of theIL-17A/F polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21(1991)).

In one approach, the three-dimensional structure of the IL-17A/Fpolypeptide, or of an IL-17A/F polypeptide-inhibitor complex, isdetermined by x-ray crystallography, by computer modeling or, mosttypically, by a combination of the two approaches. Both the shape andcharges of the IL-17A/F polypeptide must be ascertained to elucidate thestructure and to determine active site(s) of the molecule. Less often,useful information regarding the structure of the IL-17A/F polypeptidemay be gained by modeling based on the structure of homologous proteins.In both cases, relevant structural information is used to designanalogous IL-17A/F polypeptide-like molecules or to identify efficientinhibitors. Useful examples of rational drug design may includemolecules which have improved activity or stability as shown by Braxtonand Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors,agonists, or antagonists of native peptides as shown by Athauda et al.,J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the IL-17A/Fpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the IL-17A/Fpolypeptide amino acid sequence provided herein will provide guidance tothose employing computer modeling techniques in place of or in additionto x-ray crystallography.

1-22. (canceled)
 23. An isolated polypeptide having at least 80% aminoacid sequence identity to: (a) the amino acid sequence of an IL-17A/Fpolypeptide comprising SEQ ID NO:3 and SEQ ID NO:4; or (b) the aminoacid sequence of an IL-17A/F polypeptide comprising SEQ ID NO:3 and SEQID NO:4 lacking its associated signal peptides.
 24. The isolatedpolypeptide of claim 23, wherein said IL-17 A/F polypeptide comprises aheterodimeric complex comprising SEQ ID NO:3 and SEQ ID NO:4.
 25. Theisolated polypeptide of claim 24, wherein said heterodimeric complexcomprises two interchain disulfide linkages between SEQ ID NO:3 and SEQID NO:4.
 26. The isolated polypeptide of claim 23 having at least 95%amino acid sequence identity to: (a) the amino acid sequence of anIL-17A/F polypeptide comprising SEQ ID NO:3 and SEQ ID NO:4; or (b) theamino acid sequence of an IL-17A/F polypeptide comprising SEQ ID NO:3and SEQ ID NO:4 lacking its associated signal peptides.
 27. Acomposition of matter comprising (a) the isolated polypeptide of claim23, (b) an agonist of said polypeptide, (c) an antagonist of saidpolypeptide, or (d) an antibody that binds to said polypeptide, incombination with a carrier.
 28. An isolated antibody which binds to apolypeptide according to claim
 23. 29. The isolated antibody of claim28, wherein said antibody is a monoclonal antibody, which preferably hasnonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues.
 30. The isolated antibody of claim 28,wherein said antibody is a human antibody.
 31. The isolated antibody ofclaim 28, wherein said antibody is an antibody fragment, a monoclonalantibody, a single-chain antibody, or an anti-idiotypic antibody. 32.The isolated antibody of claim 31, wherein the antibody fragment orsingle-chain antibody comprises a Fab fragment selected from the groupconsisting of the amino acid sequence shown in FIG. 6 as SEQ ID NO:9,SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, wherein said Fabfragment further comprises three heavy chain variable regions containingCDR-H1 consisting of amino acid residues 7 to 16 of SEQ ID NOs:9-42,CDR-H2 consisting of amino acid residues 30 to 46 of SEQ ID NOs:9-42,and CDR-H3 consisting of amino acid residue 78 to at least amino acidresidue 96 of SEQ ID NOs:9-42, wherein said isolated Fab fragment iscapable of binding IL-17A/F.
 33. An isolated nucleic acid moleculeselected from the group consisting of the nucleotide sequence of SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, wherein said nucleicacid molecule encodes the Fab fragment shown as SEQ ID NO:9, SEQ IDNO:10; SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, or SEQ ID NO:42, wherein said Fab fragment iscapable of binding to IL-17 A/F.
 34. The composition of matter of claim27 which is useful for the treatment of an immune related disease in amammal.
 35. The composition of matter of claim 27, wherein (a), (b) or(d) is capable of (i) increasing the proliferation of T-lymphocytes in amammal, or (ii) increasing infiltration of inflammatory cells into atissue of a mammal.
 36. An article of manufacture, comprising: acontainer; a label on said container; and a composition of matteraccording to claim 27 contained within said container, wherein label onsaid container indicates that said composition of matter can be used fortreating an immune related disease.
 37. A method of treating an immunerelated disorder in a mammal in need thereof comprising administering tosaid mammal a therapeutically effective amount of (a) a polypeptide ofclaim 23, (b) an agonist of said polypeptide, (c) an antagonist of saidpolypeptide, or (d) an antibody that binds to said polypeptide.
 38. Themethod of claim 37, wherein the immune related disorder is systemiclupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenilechronic arthritis, a spondyloarthropathy, systemic sclerosis, anidiopathic inflammatory myopathy, Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renaldisease, a demyelinating disease of the central or peripheral nervoussystem, idiopathic demyelinating polyneuropathy, Guillain-Barr{acuteover (ε)} syndrome, a chronic inflammatory demyelinating polyneuropathy,a hepatobiliary disease, infectious or autoimmune chronic activehepatitis, primary biliary cirrhosis, granulomatous hepatitis,sclerosing cholangitis, inflammatory bowel disease, gluten-sensitiveenteropathy, Whipple's disease, an autoimmune or immune-mediated skindisease, a bullous skin disease, erythema multiforme, contactdermatitis, psoriasis, an allergic disease, asthma, allergic rhinitis,atopic dermatitis, food hypersensitivity, urticaria, an immunologicdisease of the lung, eosinophilic pneumonia, idiopathic pulmonaryfibrosis, hypersensitivity pneumonitis, a transplantation associateddisease, graft rejection or graft-versus-host-disease.
 39. A vectorcomprising the nucleic acid molecule of claim
 33. 40. The vector ofclaim 39 operably linked to control sequences recognized by a host celltransformed with the vector.
 41. A host cell comprising the vector ofclaim
 39. 42. The host cell of claim 41, wherein said cell is a CHOcell, an E. coli cell, a yeast cell or a Baculovirus infected insectcell.
 43. A process for producing an antibody according to claim 28comprising culturing the host cell of claim 41 under conditions suitablefor expression of said antibody and recovering said antibody from thecell culture.